Communication-dependent mineralization of osteoblasts via gap junctions.

PubMed

Hashida, Yukihiko; Nakahama, Ken-ichi; Shimizu, Kaori; Akiyama, Masako; Harada, Kiyoshi; Morita, Ikuo

2014-04-01

Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2. Copyright © 2013 Elsevier Inc. All rights reserved.

hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

PubMed Central

Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

2016-01-01

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

CoGAPS matrix factorization algorithm identifies transcriptional changes in AP-2alpha target genes in feedback from therapeutic inhibition of the EGFR network

PubMed Central

Thakar, Manjusha; Howard, Jason D.; Kagohara, Luciane T.; Krigsfeld, Gabriel; Ranaweera, Ruchira S.; Hughes, Robert M.; Perez, Jimena; Jones, Siân; Favorov, Alexander V.; Carey, Jacob; Stein-O'Brien, Genevieve; Gaykalova, Daria A.; Ochs, Michael F.; Chung, Christine H.

2016-01-01

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance. PMID:27650546

Silk Gland Gene Expression during Larval-Pupal Transition in the Cotton Leaf Roller Sylepta derogata (Lepidoptera: Pyralidae)

PubMed Central

Su, Honghua; Cheng, Yuming; Wang, Zhongyang; Li, Zhong; Stanley, David; Yang, Yizhong

2015-01-01

The cotton leaf roller, Sylepta derogata, is a silk-producing insect pest. While young larvae feed on the underside of leaves, the older ones roll cotton leaves and feed on the leaf edges, which defoliates cotton plants. The larvae produce silk to stabilize the rolled leaf and to balloon from used to new leaves. Despite the significance of silk in the biology of pest insect species, there is virtually no information on the genes involved in their silk production. This is a substantial knowledge gap because some of these genes may be valuable targets for developing molecular pest management technologies. We addressed the gap by posing the hypothesis that silk gland gene expression changes during the transition from larvae to pupae. We tested our hypothesis using RNA-seq to investigate changes in silk gland gene expression at three developmental stages, 5th instar larvae (silk producing; 15,445,926 clean reads), prepupae (reduced silk producing; 13,758,154) and pupae (beyond silk producing; 16,787,792). We recorded 60,298 unigenes and mapped 50,158 (larvae), 48,415 (prepupae) and 46,623 (pupae) of them to the NCBI database. Most differentially expressed genes in the 5th instar larvae/prepupae libraries were relevant to nucleotide synthesis and maintenance of silk gland function. We identified down-regulated transcriptional factors and several genes involved in silk formation in the three libraries and verified the expression pattern of eight genes by qPCR. The developmental- and tissue-specific expression patterns of the fibroin light chain gene showed it was highly expressed during the larval silk-producing stage. We recorded highest expression of this gene in the larval silk gland, compared to other tissues, including midgut, hindgut, epidermis, Malpighian tubes, hemolymph and fat body. These data are a genetic resource to guide selection of key genes that may be targeted for in planta and other gene-silencing technologies for sustainable cotton agriculture. PMID:26352931

Silk Gland Gene Expression during Larval-Pupal Transition in the Cotton Leaf Roller Sylepta derogata (Lepidoptera: Pyralidae).

PubMed

Su, Honghua; Cheng, Yuming; Wang, Zhongyang; Li, Zhong; Stanley, David; Yang, Yizhong

2015-01-01

The cotton leaf roller, Sylepta derogata, is a silk-producing insect pest. While young larvae feed on the underside of leaves, the older ones roll cotton leaves and feed on the leaf edges, which defoliates cotton plants. The larvae produce silk to stabilize the rolled leaf and to balloon from used to new leaves. Despite the significance of silk in the biology of pest insect species, there is virtually no information on the genes involved in their silk production. This is a substantial knowledge gap because some of these genes may be valuable targets for developing molecular pest management technologies. We addressed the gap by posing the hypothesis that silk gland gene expression changes during the transition from larvae to pupae. We tested our hypothesis using RNA-seq to investigate changes in silk gland gene expression at three developmental stages, 5th instar larvae (silk producing; 15,445,926 clean reads), prepupae (reduced silk producing; 13,758,154) and pupae (beyond silk producing; 16,787,792). We recorded 60,298 unigenes and mapped 50,158 (larvae), 48,415 (prepupae) and 46,623 (pupae) of them to the NCBI database. Most differentially expressed genes in the 5th instar larvae/prepupae libraries were relevant to nucleotide synthesis and maintenance of silk gland function. We identified down-regulated transcriptional factors and several genes involved in silk formation in the three libraries and verified the expression pattern of eight genes by qPCR. The developmental- and tissue-specific expression patterns of the fibroin light chain gene showed it was highly expressed during the larval silk-producing stage. We recorded highest expression of this gene in the larval silk gland, compared to other tissues, including midgut, hindgut, epidermis, Malpighian tubes, hemolymph and fat body. These data are a genetic resource to guide selection of key genes that may be targeted for in planta and other gene-silencing technologies for sustainable cotton agriculture.

GAP-43 Gene Expression Regulates Information Storage

ERIC Educational Resources Information Center

Holahan, Matthew R.; Honegger, Kyle S.; Tabatadze, Nino; Routtenberg, Aryeh

2007-01-01

Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at…

Efficient Reverse-Engineering of a Developmental Gene Regulatory Network

PubMed Central

Cicin-Sain, Damjan; Ashyraliyev, Maksat; Jaeger, Johannes

2012-01-01

Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms. PMID:22807664

Analysis of functional importance of binding sites in the Drosophila gap gene network model.

PubMed

Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

2015-01-01

The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling.

PubMed

Prpic, Nikola-Michael; Janssen, Ralf; Wigand, Barbara; Klingler, Martin; Damen, Wim G M

2003-12-01

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Here, we analyze appendage genes in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic (dpp) expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax (hth) and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role of this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. We comment on the significance of this finding for attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, our results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies.

Essential roles for lines in mediating leg and antennal proximodistal patterning and generating a stable Notch signaling interface at segment borders.

PubMed

Greenberg, Lina; Hatini, Victor

2009-06-01

The Drosophila leg imaginal disc provides a paradigm with which to understand the fundamental developmental mechanisms that generate an intricate appendage structure. Leg formation depends on the subdivision of the leg proximodistal (PD) axis into broad domains by the leg gap genes. The leg gap genes act combinatorially to initiate the expression of the Notch ligands Delta (Dl) and Serrate (Ser) in a segmental pattern. Dl and Ser induce the expression of a set of transcriptional regulators along the segment border, which mediate leg segment growth and joint morphogenesis. Here we show that Lines accumulates in nuclei in the presumptive tarsus and the inter-joints of proximal leg segments and governs the formation of these structures by destabilizing the nuclear protein Bowl. Across the presumptive tarsus, lines modulates the opposing expression landscapes of the leg gap gene dachshund (dac) and the tarsal PD genes, bric-a-brac 2 (bab), apterous (ap) and BarH1 (Bar). In this manner, lines inhibits proximal tarsal fates and promotes medial and distal tarsal fates. Across proximal leg segments, lines antagonizes bowl to promote Dl expression by relief-of-repression. In turn, Dl signals asymmetrically to stabilize Bowl in adjacent distal cells. Bowl, then, acts cell-autonomously, together with one or more redundant factors, to repress Dl expression. Together, lines and bowl act as a binary switch to generate a stable Notch signaling interface between Dl-expressing cells and adjacent distal cell. lines plays analogous roles in developing antennae, which are serially homologous to legs, suggesting evolutionarily conserved roles for lines in ventral appendage formation.

Evidence for Altered Ca2+ Handling in Growth Associated Protein 43-Knockout Skeletal Muscle

PubMed Central

Caprara, Giusy A.; Morabito, Caterina; Perni, Stefano; Navarra, Riccardo; Guarnieri, Simone; Mariggiò, Maria A.

2016-01-01

Neuronal growth-associated protein 43 (GAP43) has crucial roles in the nervous system, and during development, regeneration after injury, and learning and memory. GAP43 is expressed in mouse skeletal muscle fibers and satellite cells, with suggested its involvement in intracellular Ca2+ handling. However, the physiological role of GAP43 in muscle remains unknown. Using a GAP43-knockout (GAP43−/−) mouse, we have defined the role of GAP43 in skeletal muscle. GAP43−/− mice showed low survival beyond weaning, reduced adult body weight, decreased muscle strength, and changed myofiber ultrastructure, with no significant differences in the expression of markers of satellite cell and myotube progression through the myogenic program. Thus, GAP43 expression is involved in timing of muscle maturation in-vivo. Intracellular Ca2+ measurements in-vitro in myotubes revealed GAP43 involvement in Ca2+ handling. In the absence of GAP43 expression, the spontaneous Ca2+ variations had greater amplitudes and higher frequency. In GAP43−/− myotubes, also the intracellular Ca2+ variations induced by the activation of dihydropyridine and ryanodine Ca2+ channels, resulted modified. These evidences suggested dysregulation of Ca2+ homeostasis. The emerging hypothesis indicates that GAP43 interacts with calmodulin to indirectly modulate the activities of dihydropyridine and ryanodine Ca2+ channels. This thus influences intracellular Ca2+ dynamics and its related intracellular patterns, from functional excitation-contraction coupling, to cell metabolism, and gene expression. PMID:27833566

Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis.

PubMed

Kraidlova, Lucie; Schrevens, Sanne; Tournu, Hélène; Van Zeebroeck, Griet; Sychrova, Hana; Van Dijck, Patrick

2016-01-01

Amino acids are key sources of nitrogen for growth of Candida albicans . In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to Sc Gap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae , where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of Sc Sam3, the only S -adenosylmethionine (SAM) transporter in S. cerevisiae , and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of Sc Gap1 and that C. albicans Gap4 is an orthologue of Sc Sam3, an S -adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans .

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice.

PubMed

Crispino, Giulia; Di Pasquale, Giovanni; Scimemi, Pietro; Rodriguez, Laura; Galindo Ramirez, Fabian; De Siati, Romolo Daniele; Santarelli, Rosa Maria; Arslan, Edoardo; Bortolozzi, Mario; Chiorini, John A; Mammano, Fabio

2011-01-01

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

eQTL Mapping Using RNA-seq Data

PubMed Central

Hu, Yijuan

2012-01-01

As RNA-seq is replacing gene expression microarrays to assess genome-wide transcription abundance, gene expression Quantitative Trait Locus (eQTL) studies using RNA-seq have emerged. RNA-seq delivers two novel features that are important for eQTL studies. First, it provides information on allele-specific expression (ASE), which is not available from gene expression microarrays. Second, it generates unprecedentedly rich data to study RNA-isoform expression. In this paper, we review current methods for eQTL mapping using ASE and discuss some future directions. We also review existing works that use RNA-seq data to study RNA-isoform expression and we discuss the gaps between these works and isoform-specific eQTL mapping. PMID:23667399

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

PubMed Central

Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

2014-01-01

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release.

PubMed

Müller, Martin; Pym, Edward C G; Tong, Amy; Davis, Graeme W

2011-02-24

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity. Copyright © 2011 Elsevier Inc. All rights reserved.

Nerve stepping stone has minimal impact in aiding regeneration across long acellular nerve allografts.

PubMed

Yan, Ying; Hunter, Daniel A; Schellhardt, Lauren; Ee, Xueping; Snyder-Warwick, Alison K; Moore, Amy M; Mackinnon, Susan E; Wood, Matthew D

2018-02-01

Acellular nerve allografts (ANAs) yield less consistent favorable outcomes compared with autografts for long gap reconstructions. We evaluated whether a hybrid ANA can improve 6-cm gap reconstruction. Rat sciatic nerve was transected and repaired with either 6-cm hybrid or control ANAs. Hybrid ANAs were generated using a 1-cm cellular isograft between 2.5-cm ANAs, whereas control ANAs had no isograft. Outcomes were assessed by graft gene and marker expression (n = 4; at 4 weeks) and motor recovery and nerve histology (n = 10; at 20 weeks). Hybrid ANAs modified graft gene and marker expression and promoted modest axon regeneration across the 6-cm defect compared with control ANA (P < 0.05), but yielded no muscle recovery. Control ANAs had no appreciable axon regeneration across the 6-cm defect. A hybrid ANA confers minimal motor recovery benefits for regeneration across long gaps. Clinically, the authors will continue to reconstruct long nerve gaps with autografts. Muscle Nerve 57: 260-267, 2018. © 2017 Wiley Periodicals, Inc.

A Constitutive Expression System for Cellulase Secretion in Escherichia coli and Its Use in Bioethanol Production

PubMed Central

Munjal, Neha; Jawed, Kamran; Wajid, Saima; Yazdani, Syed Shams

2015-01-01

The production of biofuels from lignocellulosic biomass appears to be attractive and viable due to the abundance and availability of this biomass. The hydrolysis of this biomass, however, is challenging because of the complex lignocellulosic structure. The ability to produce hydrolytic cellulase enzymes in a cost-effective manner will certainly accelerate the process of making lignocellulosic ethanol production a commercial reality. These cellulases may need to be produced aerobically to generate large amounts of protein in a short time or anaerobically to produce biofuels from cellulose via consolidated bioprocessing. Therefore, it is important to identify a promoter that can constitutively drive the expression of cellulases under both aerobic and anaerobic conditions without the need for an inducer. Using lacZ as reporter gene, we analyzed the strength of the promoters of four genes, namely lacZ, gapA, ldhA and pflB, and found that the gapA promoter yielded the maximum expression of the β-galactosidase enzyme under both aerobic and anaerobic conditions. We further cloned the genes for two cellulolytic enzymes, β-1,4-endoglucanase and β-1,4-glucosidase, under the control of the gapA promoter, and we expressed these genes in Escherichia coli, which secreted the products into the extracellular medium. An ethanologenic E. colistrain transformed with the secretory β-glucosidase gene construct fermented cellobiose in both defined and complex medium. This recombinant strain also fermented wheat straw hydrolysate containing glucose, xylose and cellobiose into ethanol with an 85% efficiency of biotransformation. An ethanologenic strain that constitutively secretes a cellulolytic enzyme is a promising platform for producing lignocellulosic ethanol. PMID:25768292

Filling gaps in PPAR-alpha signaling through comparative nutrigenomics analysis.

PubMed

Cavalieri, Duccio; Calura, Enrica; Romualdi, Chiara; Marchi, Emmanuela; Radonjic, Marijana; Van Ommen, Ben; Müller, Michael

2009-12-11

The application of high-throughput genomic tools in nutrition research is a widespread practice. However, it is becoming increasingly clear that the outcome of individual expression studies is insufficient for the comprehensive understanding of such a complex field. Currently, the availability of the large amounts of expression data in public repositories has opened up new challenges on microarray data analyses. We have focused on PPARalpha, a ligand-activated transcription factor functioning as fatty acid sensor controlling the gene expression regulation of a large set of genes in various metabolic organs such as liver, small intestine or heart. The function of PPARalpha is strictly connected to the function of its target genes and, although many of these have already been identified, major elements of its physiological function remain to be uncovered. To further investigate the function of PPARalpha, we have applied a cross-species meta-analysis approach to integrate sixteen microarray datasets studying high fat diet and PPARalpha signal perturbations in different organisms. We identified 164 genes (MDEGs) that were differentially expressed in a constant way in response to a high fat diet or to perturbations in PPARs signalling. In particular, we found five genes in yeast which were highly conserved and homologous of PPARalpha targets in mammals, potential candidates to be used as models for the equivalent mammalian genes. Moreover, a screening of the MDEGs for all known transcription factor binding sites and the comparison with a human genome-wide screening of Peroxisome Proliferating Response Elements (PPRE), enabled us to identify, 20 new potential candidate genes that show, both binding site, both change in expression in the condition studied. Lastly, we found a non random localization of the differentially expressed genes in the genome. The results presented are potentially of great interest to resume the currently available expression data, exploiting the power of in silico analysis filtered by evolutionary conservation. The analysis enabled us to indicate potential gene candidates that could fill in the gaps with regards to the signalling of PPARalpha and, moreover, the non-random localization of the differentially expressed genes in the genome, suggest that epigenetic mechanisms are of importance in the regulation of the transcription operated by PPARalpha.

Analyses of interactions among pair-rule genes and the gap gene Krüppel in Bombyx segmentation.

PubMed

Nakao, Hajime

2015-09-01

In the short-germ insect Tribolium, a pair-rule gene circuit consisting of the Tribolium homologs of even-skipped, runt, and odd-skipped (Tc-eve, Tc-run and Tc-odd, respectively) has been implicated in segment formation. To examine the application of the model to other taxa, I studied the expression and function of pair-rule genes in Bombyx mori, together with a Bombyx homolog of Krüppel (Bm-Kr), a known gap gene. Knockdown embryos of Bombyx homologs of eve, run and odd (Bm-eve, Bm-run and Bm-odd) exhibited asegmental phenotypes similar to those of Tribolium knockdowns. However, pair-rule gene interactions were similar to those of both Tribolium and Drosophila, which, different from Tribolium, shows a hierarchical segmentation mode. Additionally, the Bm-odd expression pattern shares characteristics with those of Drosophila pair-rule genes that receive upstream regulatory input. On the other hand, Bm-Kr knockdowns exhibited a large posterior segment deletion as observed in short-germ insects. However, a detailed analysis of these embryos indicated that Bm-Kr modulates expression of pair-rule genes like in Drosophila, although the mechanisms appear to be different. This suggested hierarchical interactions between Bm-Kr and pair-rule genes. Based on these results, I concluded that the pair-rule gene circuit model that describes Tribolium development is not applicable to Bombyx. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell-specific prediction and application of drug-induced gene expression profiles.

PubMed

Hodos, Rachel; Zhang, Ping; Lee, Hao-Chih; Duan, Qiaonan; Wang, Zichen; Clark, Neil R; Ma'ayan, Avi; Wang, Fei; Kidd, Brian; Hu, Jianying; Sontag, David; Dudley, Joel

2018-01-01

Gene expression profiling of in vitro drug perturbations is useful for many biomedical discovery applications including drug repurposing and elucidation of drug mechanisms. However, limited data availability across cell types has hindered our capacity to leverage or explore the cell-specificity of these perturbations. While recent efforts have generated a large number of drug perturbation profiles across a variety of human cell types, many gaps remain in this combinatorial drug-cell space. Hence, we asked whether it is possible to fill these gaps by predicting cell-specific drug perturbation profiles using available expression data from related conditions--i.e. from other drugs and cell types. We developed a computational framework that first arranges existing profiles into a three-dimensional array (or tensor) indexed by drugs, genes, and cell types, and then uses either local (nearest-neighbors) or global (tensor completion) information to predict unmeasured profiles. We evaluate prediction accuracy using a variety of metrics, and find that the two methods have complementary performance, each superior in different regions in the drug-cell space. Predictions achieve correlations of 0.68 with true values, and maintain accurate differentially expressed genes (AUC 0.81). Finally, we demonstrate that the predicted profiles add value for making downstream associations with drug targets and therapeutic classes.

Cell-specific prediction and application of drug-induced gene expression profiles

PubMed Central

Hodos, Rachel; Zhang, Ping; Lee, Hao-Chih; Duan, Qiaonan; Wang, Zichen; Clark, Neil R.; Ma'ayan, Avi; Wang, Fei; Kidd, Brian; Hu, Jianying; Sontag, David

2017-01-01

Gene expression profiling of in vitro drug perturbations is useful for many biomedical discovery applications including drug repurposing and elucidation of drug mechanisms. However, limited data availability across cell types has hindered our capacity to leverage or explore the cell-specificity of these perturbations. While recent efforts have generated a large number of drug perturbation profiles across a variety of human cell types, many gaps remain in this combinatorial drug-cell space. Hence, we asked whether it is possible to fill these gaps by predicting cell-specific drug perturbation profiles using available expression data from related conditions--i.e. from other drugs and cell types. We developed a computational framework that first arranges existing profiles into a three-dimensional array (or tensor) indexed by drugs, genes, and cell types, and then uses either local (nearest-neighbors) or global (tensor completion) information to predict unmeasured profiles. We evaluate prediction accuracy using a variety of metrics, and find that the two methods have complementary performance, each superior in different regions in the drug-cell space. Predictions achieve correlations of 0.68 with true values, and maintain accurate differentially expressed genes (AUC 0.81). Finally, we demonstrate that the predicted profiles add value for making downstream associations with drug targets and therapeutic classes. PMID:29218867

Spatially coordinated dynamic gene transcription in living pituitary tissue

PubMed Central

Featherstone, Karen; Hey, Kirsty; Momiji, Hiroshi; McNamara, Anne V; Patist, Amanda L; Woodburn, Joanna; Spiller, David G; Christian, Helen C; McNeilly, Alan S; Mullins, John J; Finkenstädt, Bärbel F; Rand, David A; White, Michael RH; Davis, Julian RE

2016-01-01

Transcription at individual genes in single cells is often pulsatile and stochastic. A key question emerges regarding how this behaviour contributes to tissue phenotype, but it has been a challenge to quantitatively analyse this in living cells over time, as opposed to studying snap-shots of gene expression state. We have used imaging of reporter gene expression to track transcription in living pituitary tissue. We integrated live-cell imaging data with statistical modelling for quantitative real-time estimation of the timing of switching between transcriptional states across a whole tissue. Multiple levels of transcription rate were identified, indicating that gene expression is not a simple binary ‘on-off’ process. Immature tissue displayed shorter durations of high-expressing states than the adult. In adult pituitary tissue, direct cell contacts involving gap junctions allowed local spatial coordination of prolactin gene expression. Our findings identify how heterogeneous transcriptional dynamics of single cells may contribute to overall tissue behaviour. DOI: http://dx.doi.org/10.7554/eLife.08494.001 PMID:26828110

Early diffusion of gene expression profiling in breast cancer patients associated with areas of high income inequality.

PubMed

Ponce, Ninez A; Ko, Michelle; Liang, Su-Ying; Armstrong, Joanne; Toscano, Michele; Chanfreau-Coffinier, Catherine; Haas, Jennifer S

2015-04-01

With the Affordable Care Act reducing coverage disparities, social factors could prominently determine where and for whom innovations first diffuse in health care markets. Gene expression profiling is a potentially cost-effective innovation that guides chemotherapy decisions in early-stage breast cancer, but adoption has been uneven across the United States. Using a sample of commercially insured women, we evaluated whether income inequality in metropolitan areas was associated with receipt of gene expression profiling during its initial diffusion in 2006-07. In areas with high income inequality, gene expression profiling receipt was higher than elsewhere, but it was associated with a 10.6-percentage-point gap between high- and low-income women. In areas with low rates of income inequality, gene expression profiling receipt was lower, with no significant differences by income. Even among insured women, income inequality may indirectly shape diffusion of gene expression profiling, with benefits accruing to the highest-income patients in the most unequal places. Policies reducing gene expression profiling disparities should address low-inequality areas and, in unequal places, practice settings serving low-income patients. Project HOPE—The People-to-People Health Foundation, Inc.

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

NASA Astrophysics Data System (ADS)

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.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

PubMed

Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

1998-01-08

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Coregulation of srGAP1 by Wnt and Androgen Receptor Signaling: A New Target for Treatment of CRPC

DTIC Science & Technology

2015-10-01

Specific Aim 1: Test the... Specific Aim2: Test the hypothesis that down regulating srGAP1 in CRPC cells change phenotypic...direct interaction between AR and β-catenin seemed to elicit a specific expression of a set of target genes in low androgen conditions in CRPC.

Filling gaps in PPAR-alpha signaling through comparative nutrigenomics analysis

PubMed Central

2009-01-01

Background The application of high-throughput genomic tools in nutrition research is a widespread practice. However, it is becoming increasingly clear that the outcome of individual expression studies is insufficient for the comprehensive understanding of such a complex field. Currently, the availability of the large amounts of expression data in public repositories has opened up new challenges on microarray data analyses. We have focused on PPARα, a ligand-activated transcription factor functioning as fatty acid sensor controlling the gene expression regulation of a large set of genes in various metabolic organs such as liver, small intestine or heart. The function of PPARα is strictly connected to the function of its target genes and, although many of these have already been identified, major elements of its physiological function remain to be uncovered. To further investigate the function of PPARα, we have applied a cross-species meta-analysis approach to integrate sixteen microarray datasets studying high fat diet and PPARα signal perturbations in different organisms. Results We identified 164 genes (MDEGs) that were differentially expressed in a constant way in response to a high fat diet or to perturbations in PPARs signalling. In particular, we found five genes in yeast which were highly conserved and homologous of PPARα targets in mammals, potential candidates to be used as models for the equivalent mammalian genes. Moreover, a screening of the MDEGs for all known transcription factor binding sites and the comparison with a human genome-wide screening of Peroxisome Proliferating Response Elements (PPRE), enabled us to identify, 20 new potential candidate genes that show, both binding site, both change in expression in the condition studied. Lastly, we found a non random localization of the differentially expressed genes in the genome. Conclusion The results presented are potentially of great interest to resume the currently available expression data, exploiting the power of in silico analysis filtered by evolutionary conservation. The analysis enabled us to indicate potential gene candidates that could fill in the gaps with regards to the signalling of PPARα and, moreover, the non-random localization of the differentially expressed genes in the genome, suggest that epigenetic mechanisms are of importance in the regulation of the transcription operated by PPARα. PMID:20003344

Course 10: Three Lectures on Biological Networks

NASA Astrophysics Data System (ADS)

Magnasco, M. O.

1 Enzymatic networks. Proofreading knots: How DNA topoisomerases disentangle DNA 1.1 Length scales and energy scales 1.2 DNA topology 1.3 Topoisomerases 1.4 Knots and supercoils 1.5 Topological equilibrium 1.6 Can topoisomerases recognize topology? 1.7 Proposal: Kinetic proofreading 1.8 How to do it twice 1.9 The care and proofreading of knots 1.10 Suppression of supercoils 1.11 Problems and outlook 1.12 Disquisition 2 Gene expression networks. Methods for analysis of DNA chip experiments 2.1 The regulation of gene expression 2.2 Gene expression arrays 2.3 Analysis of array data 2.4 Some simplifying assumptions 2.5 Probeset analysis 2.6 Discussion 3 Neural and gene expression networks: Song-induced gene expression in the canary brain 3.1 The study of songbirds 3.2 Canary song 3.3 ZENK 3.4 The blush 3.5 Histological analysis 3.6 Natural vs. artificial 3.7 The Blush II: gAP 3.8 Meditation

Connexin32 expression in central and peripheral nervous systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deschenes, S.M.; Scherer, S.S.; Fischbeck, K.H.

1994-09-01

Mutations have been identified in the gap junction gene, connexin32 (Cx32), in patients affected with the X-linked form of the demyelinating neuropathy, Charcot-Marie-Tooth disease (CMTX). Gap junctions composed of Cx32 are present and developmentally regulated in a wide variety of tissues. In peripheral nerve, our immunohistochemical analysis localized Cx32 to the noncompacted myelin of the paranodal regions and the Schmidt-Lantermann incisures, where previous studies describe gap junctions. In contrast to the location of Cx32 in peripheral nerve and the usual restriction of clinical manifestations to the peripheral nervous system (PNS) (abstract by Paulson describes an exception), preliminary studies show thatmore » Cx32 is present in the compacted myelin of the central nervous system (CNS), as demonstrated by radial staining through the myelin sheath of oligodendrocytes in rat spinal cord. Analysis of Cx32 expression in various regions of rat CNS during development shows that the amount of Cx32 mRNA and protein increases as myelination increases, a pattern observed for other myelin genes. Studies in the PNS provide additional evidence that Cx32 and myelin genes are coordinately regulated at the transcriptional level; Cx32 and peripheral myelin gene PMP-22 mRNAs are expressed in parallel following transient or permanent nerve injury. Differences in post-translational regulation of Cx32 in the CNS and PNS may be indicated by the presence of a faster migrating form of Cs32 in cerebrum versus peripheral nerve. Studies are currently underway to determine the unique role of Cx32 in peripheral nerve.« less

TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

PubMed

Tacheau, Charlotte; Fontaine, Juliette; Loy, Jennifer; Mauviel, Alain; Verrecchia, Franck

2008-12-01

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Drosophila segmentation: supercomputer simulation of prepattern hierarchy.

PubMed

Hunding, A; Kauffman, S A; Goodwin, B C

1990-08-09

Spontaneous prepattern formation in a two level hierarchy of reaction-diffusion systems is simulated in three space co-ordinates and time, mimicking gap gene and primary pair-rule gene expression. The model rests on the idea of Turing systems of the second kind, in which one prepattern generates position dependent rate constants for a subsequent reaction-diffusion system. Maternal genes are assumed responsible for setting up gradients from the anterior and posterior ends, one of which is needed to stabilize a double period prepattern suggested to underly the read out of the gap genes. The resulting double period pattern in turn stabilizes the next prepattern in the hierarchy, which has a short wavelength with many characteristics of the stripes seen in actual primary pair-rule gene expression. Without such hierarchical stabilization, reaction-diffusion mechanisms yield highly patchy short wave length patterns, and thus unreliable stripes. The model yields seven stable stripes located in the middle of the embryo, with the potential for additional expression near the poles, as observed experimentally. The model does not rely on specific chemical reaction kinetics, rather the effect is general to many such kinetic schemes. This makes it robust to parameter changes, and it has good potential for adapting to size and shape changes as well. The study thus suggests that the crucial organizing principle in early Drosophila embryogenesis is based on global field mechanisms, not on particular local interactions.

Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes

PubMed Central

Matsuoka, Yuji; Bando, Tetsuya; Watanabe, Takahito; Ishimaru, Yoshiyasu; Noji, Sumihare; Popadić, Aleksandar; Mito, Taro

2015-01-01

In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. PMID:25948756

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

PubMed Central

Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

2005-01-01

More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila.

PubMed

Verd, Berta; Clark, Erik; Wotton, Karl R; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton; Jaeger, Johannes

2018-02-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila

PubMed Central

Verd, Berta; Clark, Erik; Wotton, Karl R.; Janssens, Hilde; Jiménez-Guri, Eva; Crombach, Anton

2018-01-01

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects. PMID:29451884

Genetic and functional analysis of the gene encoding GAP-43 in schizophrenia.

PubMed

Shen, Yu-Chih; Tsai, Ho-Min; Cheng, Min-Chih; Hsu, Shih-Hsin; Chen, Shih-Fen; Chen, Chia-Hsiang

2012-02-01

In earlier reports, growth-associated protein 43 (GAP-43) has been shown to be critical for initial establishment or reorganization of synaptic connections, a process thought to be disrupted in schizophrenia. Additionally, abnormal GAP-43 expression in different brain regions has been linked to this disorder in postmortem brain studies. In this study, we investigated the involvement of the gene encoding GAP-43 in the susceptibility to schizophrenia. We searched for genetic variants in the promoter region and 3 exons (including both UTR ends) of the GAP-43 gene using direct sequencing in a sample of patients with schizophrenia (n=586) and non-psychotic controls (n=576), both being Han Chinese from Taiwan, and conducted an association and functional study. We identified 11 common polymorphisms in the GAP-43 gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. Additionally, we identified 4 rare variants in 5 out of 586 patients, including 1 variant located at the promoter region (c.-258-4722G>T) and 1 synonymous (V110V) and 2 missense (G150R and P188L) variants located at exon 2. No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated that the regulatory activity of construct containing c.-258-4722T was significantly lower as compared to the wild type construct (c.-258-4722G; p<0.001). In silico analysis also demonstrated the functional relevance of other rare variants. Our study lends support to the hypothesis of multiple rare mutations in schizophrenia, and it provides genetic clues that indicate the involvement of GAP-43 in this disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis–associated glomerular injury

PubMed Central

Potla, Uma; Ni, Jie; Vadaparampil, Justin; Yang, Guozhe; Leventhal, Jeremy S.; Campbell, Kirk N.; Chuang, Peter Y.; Morozov, Alexei; He, John C.; D’Agati, Vivette D.; Klotman, Paul E.; Kaufman, Lewis

2014-01-01

Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated β1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained β1 integrin–mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of β1 integrin. PMID:24642466

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes

PubMed Central

Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J; Johnson, Anthony; Jacot, Jeffrey G

2013-01-01

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC. PMID:23634988

The basic helix-loop-helix differentiation factor Nex1/MATH-2 functions as a key activator of the GAP-43 gene

PubMed Central

Uittenbogaard, Martine; Martinka, Debra L.; Chiaramello, Anne

2006-01-01

Nex1/MATH-2 is a neurogenic basic Helix-Loop-Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP-43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP-43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP-43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP-43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP-43 promoter. The Nex1-mediated activity is restricted to the conserved E1–E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site-directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E-box is a high affinity binding site. We further found that Nex1 released the ME1 E-protein-mediated repression in a concentration dependent manner. Thus, the E1–E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N-terminal and C-terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP-43 promoter and cellular environment. PMID:12562512

Calreticulin Induces Dilated Cardiomyopathy

PubMed Central

Lee, Dukgyu; Oka, Tatsujiro; Hunter, Beth; Robinson, Alison; Papp, Sylvia; Nakamura, Kimitoshi; Srisakuldee, Wattamon; Nickel, Barbara E.; Light, Peter E.; Dyck, Jason R. B.; Lopaschuk, Gary D.; Kardami, Elissavet; Opas, Michal; Michalak, Marek

2013-01-01

Background Calreticulin, a Ca2+-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart. Methodology/Principal Findings We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca2+ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca2+-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca2+-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin. Conclusions/Significance We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca2+ handling genes, gap junction components and left ventricle remodeling. PMID:23437120

Sequential and combinatorial roles of maf family genes define proper lens development.

PubMed

Reza, Hasan Mahmud; Urano, Atsuyo; Shimada, Naoko; Yasuda, Kunio

2007-01-16

Maf proteins have been shown to play pivotal roles in lens development in vertebrates. The developing chick lens expresses at least three large Maf proteins. However, the transcriptional relationship among the three large maf genes and their various roles in transactivating the downstream genes largely remain to be elucidated. Chick embryos were electroporated with wild-type L-maf, c-maf, and mafB by in ovo electroporation, and their effects on gene expression were determined by in situ hybridization using specific probes or by immunostaining. Endogenous gene expression was determined using nonelectroporated samples. A regulation mechanism exists among the members of maf family gene. An early-expressed member of this gene family typically stimulates the expression of later-expressed members. We also examined the regulation of various lens-expressing genes with a focus on the interaction between different Maf proteins. We found that the transcriptional ability of Maf proteins varies, even when the target is the same, in parallel with their discrete functions. L-Maf and c-Maf have no effect on E-cadherin expression, whereas MafB enhances its expression and thereby impedes lens vesicle formation. This study also revealed that Maf proteins can regulate the expression of gap junction genes, connexins, and their interacting partner, major intrinsic protein (MIP), during lens development. Misexpression of L-Maf and c-Maf induces ectopic expression of Cx43 and MIP; in contrast, MafB appears to have no effect on Cx43, but induces MIP significantly as evidenced from our gain-of-function experiments. Our results indicate that large Maf function is indispensable for chick lens initiation and development. In addition, L-Maf positively regulates most of the essential genes in this program and directs a series of molecular events leading to proper formation of the lens.

Genetic Screening in C. Elegans Identifies Rho-GTPAse Activating Protein 6 as Novel HERG Regulator

PubMed Central

Potet, Franck; Petersen, Christina I.; Boutaud, Olivier; Shuai, Wen; Stepanovic, Svetlana Z.; Balser, Jeffrey R.; Kupershmidt, Sabina

2009-01-01

The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of IKr, a K+ current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that IKr recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53 % enhancement of IKr. ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG. PMID:19038263

Connexin mutations in X-linked Charcot-Marie-Tooth disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergoffen, J.; Scherer, S.S.; Wang, S.

1993-12-24

X-linked Charcot-Marie-Tooth disease (CMTX) is a form of hereditary neuropathy with demyelination. Recently, this disorder was mapped to chromosome Xq13.1. The gene for the gap junction protein connexin32 is located in the same chromosomal segment, which led to its consideration as a candidate gene for CMTX. With the use of Northern (RNA) blot and immunohistochemistry techniques, it was found that connexin32 is normally expressed in myelinated peripheral nerve. Direct sequencing of the connexin32 gene showed seven different mutations in affected persons from eight CMTX families. These findings, a demonstration of inherited defects in a gap junction protein, suggest that connexin32more » plays an important role in peripheral nerve.« less

RAS oncogene-mediated deregulation of the transcriptome: from molecular signature to function.

PubMed

Schäfer, Reinhold; Sers, Christine

2011-01-01

Transcriptome analysis of cancer cells has developed into a standard procedure to elucidate multiple features of the malignant process and to link gene expression to clinical properties. Gene expression profiling based on microarrays provides essentially correlative information and needs to be transferred to the functional level in order to understand the activity and contribution of individual genes or sets of genes as elements of the gene signature. To date, there exist significant gaps in the functional understanding of gene expression profiles. Moreover, the processes that drive the profound transcriptional alterations that characterize cancer cells remain mainly elusive. We have used pathway-restricted gene expression profiles derived from RAS oncogene-transformed cells and from RAS-expressing cancer cells to identify regulators downstream of the MAPK pathway.We describe the role of epigenetic regulation exemplified by the control of several immune genes in generic cell lines and colorectal cancer cells, particularly the functional interaction between signaling and DNA methylation. Moreover, we assess the role of the architectural transcription factor high mobility AT-hook 2 (HMGA2) as a regulator of the RAS-responsive transcriptome in ovarian epithelial cells. Finally, we describe an integrated approach combining pathway interference in colorectal cancer cells, gene expression profiling and computational analysis of regulatory elements of deregulated target genes. This strategy resulted in the identification of Y-box binding protein 1 (YBX1) as a regulator of MAPK-dependent proliferation and gene expression. The implications for a therapeutic application of HMGA2 gene silencing and the role of YBX1 as a prognostic factor are discussed.

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

PubMed Central

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

PubMed

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.).

PubMed

Lipka, A; Paukszto, L; Majewska, M; Jastrzebski, J P; Myszczynski, K; Panasiewicz, G; Szafranska, B

2017-09-01

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α 7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α 1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity.

PubMed

Jaken, Robby J; van Gorp, Sebastiaan; Joosten, Elbert A; Losen, Mario; Martínez-Martínez, Pilar; De Baets, Marc; Marcus, Marco A; Deumens, Ronald

2011-12-01

Structural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. In the present study, the spatiotemporal expression of growth-associated protein-43 (GAP-43), a protein which has been traditionally implicated in nerve fiber growth and sprouting, was investigated in relation to mechanical pain hypersensitivity. An L5 spinal nerve transection model was validated by the presence of mechanical pain hypersensitivity and an increase in the early neuronal activation marker cFos within the superficial spinal dorsal horn upon innocuous hindpaw stimulation. Spinal GAP-43 was found to be upregulated in the superficial L5 dorsal horn from 5 up to 10 days after injury. GAP-43 was co-localized with calcitonin-gene related peptide (CGRP), but not vesicular glutamate transporter-1 (VGLUT-1), IB4, or protein kinase-γ (PKC-γ), suggesting the regulation of GAP-43 in peptidergic nociceptive afferents. These GAP-43/CGRP fibers may be indicative of sprouting peptidergic fibers. Fiber sprouting largely depends on growth factors, which are typically associated with neuro-inflammatory processes. The putative role of neuropathy-induced GAP-43 expression in the development of mechanical pain hypersensitivity was investigated using the immune modulator propentofylline. Propentofylline treatment strongly attenuated the development of mechanical pain hypersensitivity and glial responses to nerve injury as measured by microglial and astroglial markers, but did not affect neuropathy-induced levels of spinal GAP-43 or GAP-43 regulation in CGRP fibers. We conclude that nerve injury induces structural plasticity in fibers expressing CGRP, which is regarded as a main player in central sensitization. Our data do not, however, support a major role of these structural changes in the onset of mechanical pain hypersensitivity.

Intra and Interspecific Variations of Gene Expression Levels in Yeast Are Largely Neutral: (Nei Lecture, SMBE 2016, Gold Coast).

PubMed

Yang, Jian-Rong; Maclean, Calum J; Park, Chungoo; Zhao, Huabin; Zhang, Jianzhi

2017-09-01

It is commonly, although not universally, accepted that most intra and interspecific genome sequence variations are more or less neutral, whereas a large fraction of organism-level phenotypic variations are adaptive. Gene expression levels are molecular phenotypes that bridge the gap between genotypes and corresponding organism-level phenotypes. Yet, it is unknown whether natural variations in gene expression levels are mostly neutral or adaptive. Here we address this fundamental question by genome-wide profiling and comparison of gene expression levels in nine yeast strains belonging to three closely related Saccharomyces species and originating from five different ecological environments. We find that the transcriptome-based clustering of the nine strains approximates the genome sequence-based phylogeny irrespective of their ecological environments. Remarkably, only ∼0.5% of genes exhibit similar expression levels among strains from a common ecological environment, no greater than that among strains with comparable phylogenetic relationships but different environments. These and other observations strongly suggest that most intra and interspecific variations in yeast gene expression levels result from the accumulation of random mutations rather than environmental adaptations. This finding has profound implications for understanding the driving force of gene expression evolution, genetic basis of phenotypic adaptation, and general role of stochasticity in evolution. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Enhancing GTEx by bridging the gaps between genotype, gene expression, and disease.

PubMed

2017-12-01

Genetic variants have been associated with myriad molecular phenotypes that provide new insight into the range of mechanisms underlying genetic traits and diseases. Identifying any particular genetic variant's cascade of effects, from molecule to individual, requires assaying multiple layers of molecular complexity. We introduce the Enhancing GTEx (eGTEx) project that extends the GTEx project to combine gene expression with additional intermediate molecular measurements on the same tissues to provide a resource for studying how genetic differences cascade through molecular phenotypes to impact human health.

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes

PubMed Central

Recouvreux, María Sol; Grasso, Esteban Nicolás; Echeverria, Pablo Christian; Rocha-Viegas, Luciana; Castilla, Lucio Hernán; Schere-Levy, Carolina; Tocci, Johanna Melisa; Kordon, Edith Claudia; Rubinstein, Natalia

2016-01-01

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability. PMID:26735887

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes.

PubMed

Recouvreux, María Sol; Grasso, Esteban Nicolás; Echeverria, Pablo Christian; Rocha-Viegas, Luciana; Castilla, Lucio Hernán; Schere-Levy, Carolina; Tocci, Johanna Melisa; Kordon, Edith Claudia; Rubinstein, Natalia

2016-02-09

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability.

Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression.

PubMed

Agrawal, Deepak K; Tang, Xun; Westbrook, Alexandra; Marshall, Ryan; Maxwell, Colin S; Lucks, Julius; Noireaux, Vincent; Beisel, Chase L; Dunlop, Mary J; Franco, Elisa

2018-05-08

Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.

Effects of age, sex, and genotype on high-sensitivity metabolomic profiles in the fruit fly, Drosophila melanogaster

PubMed Central

Hoffman, Jessica M; Soltow, Quinlyn A; Li, Shuzhao; Sidik, Alfire; Jones, Dean P; Promislow, Daniel E L

2014-01-01

Researchers have used whole-genome sequencing and gene expression profiling to identify genes associated with age, in the hope of understanding the underlying mechanisms of senescence. But there is a substantial gap from variation in gene sequences and expression levels to variation in age or life expectancy. In an attempt to bridge this gap, here we describe the effects of age, sex, genotype, and their interactions on high-sensitivity metabolomic profiles in the fruit fly, Drosophila melanogaster. Among the 6800 features analyzed, we found that over one-quarter of all metabolites were significantly associated with age, sex, genotype, or their interactions, and multivariate analysis shows that individual metabolomic profiles are highly predictive of these traits. Using a metabolomic equivalent of gene set enrichment analysis, we identified numerous metabolic pathways that were enriched among metabolites associated with age, sex, and genotype, including pathways involving sugar and glycerophospholipid metabolism, neurotransmitters, amino acids, and the carnitine shuttle. Our results suggest that high-sensitivity metabolomic studies have excellent potential not only to reveal mechanisms that lead to senescence, but also to help us understand differences in patterns of aging among genotypes and between males and females. PMID:24636523

Precision matters for position decoding in the early fly embryo

NASA Astrophysics Data System (ADS)

Petkova, Mariela D.; Tkacik, Gasper; Wieschaus, Eric F.; Bialek, William; Gregor, Thomas

Genetic networks can determine cell fates in multicellular organisms with precision that often reaches the physical limits of the system. However, it is unclear how the organism uses this precision and whether it has biological content. Here we address this question in the developing fly embryo, in which a genetic network of patterning genes reaches 1% precision in positioning cells along the embryo axis. The network consists of three interconnected layers: an input layer of maternal gradients, a processing layer of gap genes, and an output layer of pair-rule genes with seven-striped patterns. From measurements of gap gene protein expression in hundreds of wild-type embryos we construct a ``decoder'', which is a look-up table that determines cellular positions from the concentration means, variances and co-variances. When we apply the decoder to measurements in mutant embryos lacking various combinations of the maternal inputs, we predict quantitative changes in the output layer such as missing, altered or displaced stripes. We confirm these predictions by measuring pair-rule expression in the mutant embryos. Our results thereby show that the precision of the patterning network is biologically meaningful and a necessary feature for decoding cell positions in the early fly embryo.

Characterization of candidate genes in inflammatory bowel disease–associated risk loci

PubMed Central

Peloquin, Joanna M.; Sartor, R. Balfour; Newberry, Rodney D.; McGovern, Dermot P.; Yajnik, Vijay; Lira, Sergio A.

2016-01-01

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn’s disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis. PMID:27668286

Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

PubMed Central

Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

2017-01-01

Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

Too much data, but little inter-changeability: a lesson learned from mining public data on tissue specificity of gene expression.

PubMed

Li, Shuyu; Li, Yiqun Helen; Wei, Tao; Su, Eric Wen; Duffin, Kevin; Liao, Birong

2006-10-25

The tissue expression pattern of a gene often provides an important clue to its potential role in a biological process. A vast amount of gene expression data have been and are being accumulated in public repository through different technology platforms. However, exploitations of these rich data sources remain limited in part due to issues of technology standardization. Our objective is to test the data comparability between SAGE and microarray technologies, through examining the expression pattern of genes under normal physiological states across variety of tissues. There are 42-54% of genes showing significant correlations in tissue expression patterns between SAGE and GeneChip, with 30-40% of genes whose expression patterns are positively correlated and 10-15% of genes whose expression patterns are negatively correlated at a statistically significant level (p = 0.05). Our analysis suggests that the discrepancy on the expression patterns derived from technology platforms is not likely from the heterogeneity of tissues used in these technologies, or other spurious correlations resulting from microarray probe design, abundance of genes, or gene function. The discrepancy can be partially explained by errors in the original assignment of SAGE tags to genes due to the evolution of sequence databases. In addition, sequence analysis has indicated that many SAGE tags and Affymetrix array probe sets are mapped to different splice variants or different sequence regions although they represent the same gene, which also contributes to the observed discrepancies between SAGE and array expression data. To our knowledge, this is the first report attempting to mine gene expression patterns across tissues using public data from different technology platforms. Unlike previous similar studies that only demonstrated the discrepancies between the two gene expression platforms, we carried out in-depth analysis to further investigate the cause for such discrepancies. Our study shows that the exploitation of rich public expression resource requires extensive knowledge about the technologies, and experiment. Informatic methodologies for better interoperability among platforms still remain a gap. One of the areas that can be improved practically is the accurate sequence mapping of SAGE tags and array probes to full-length genes.

Associations of chemo- and radio-resistant phenotypes with the gap junction, adhesion and extracellular matrix in a three-dimensional culture model of soft sarcoma.

PubMed

Bai, Chujie; Yang, Min; Fan, Zhengfu; Li, Shu; Gao, Tian; Fang, Zhiwei

2015-06-10

Three-dimensional (3D) culture models are considered to recapitulate the cell microenvironment in solid tumors, including the extracellular matrix (ECM), cell-cell interactions, and signal transduction. These functions are highly correlated with cellular behaviors and contribute to resistances against chemo- and radio-therapies. However, the biochemical effects and mechanisms remain unknown in soft sarcoma. Therefore, we developed an in vitro 3D model of sarcoma to analyze the reasons of the chemo- and radio-resistance in therapies. Four soft sarcoma cell lines, HT1080, RD, SW872, and human osteosarcoma cell line 1 (HOSS1), a cell line established from a patient-derived xenograft, were applied to 3D culture and treated with growth factors in methylcellulose-containing medium. Spheroids were examined morphologically and by western blotting, RT-qPCR, and immunofluorescence staining to analyze cell adhesion, gap junctions, ECM genes, and related factors. Proliferation and colony formation assays were performed to assess chemo- and radio-resistances between 3D and two-dimensional (2D) cell cultures. Annexin V and Propidium Iodide staining was used to detect early apoptotic sarcoma cells treated with Doxorubicin, Gemcitabine, and Docetaxel in the 3D model. The four soft sarcoma cell lines formed spheres in vitro by culture in modified condition medium. Compared with 2D cell culture, expression of ECM genes and proteins, including COL1A1, LOX, SED1, FN1, and LAMA4, was significantly increased in 3D culture. Analysis of cadherin and gap junction molecules showed significant changes in the gene and protein expression profiles under 3D conditions. These changes affected cell-cell communication and were mainly associated with biological processes such as cell proliferation and apoptosis related to chemo- and radio-resistances. Our findings revealed significant differences between 3D and 2D cell culture systems, and indicated that cellular responsiveness to external stress such as radiation and chemotherapeutics is influenced by differential expression of genes and proteins involved in regulation of the ECM, cell adhesion, and gap junction signaling.

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

PubMed

Li, Yong-Fang; Mahalingam, Ramamurthy; Sunkar, Ramanjulu

2017-01-01

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

PubMed Central

Keith, Brian J.; Jozwiakowski, Stanislaw K.; Connolly, Bernard A.

2013-01-01

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated–naphthoylated DEAE–cellulose, resulting in a low background mutation frequency (∼1 × 10−4). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system. PMID:23098700

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Heterogeneous conservation of Dlx paralog co-expression in jawed vertebrates.

PubMed

Debiais-Thibaud, Mélanie; Metcalfe, Cushla J; Pollack, Jacob; Germon, Isabelle; Ekker, Marc; Depew, Michael; Laurenti, Patrick; Borday-Birraux, Véronique; Casane, Didier

2013-01-01

The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model. The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family. Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Medium-throughput processing of whole mount in situ hybridisation experiments into gene expression domains.

PubMed

Crombach, Anton; Cicin-Sain, Damjan; Wotton, Karl R; Jaeger, Johannes

2012-01-01

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, "medium-throughput" pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.

Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

PubMed Central

García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

2015-01-01

Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

The segment polarity network is a robust developmental module

NASA Astrophysics Data System (ADS)

von Dassow, George; Meir, Eli; Munro, Edwin M.; Odell, Garrett M.

2000-07-01

All insects possess homologous segments, but segment specification differs radically among insect orders. In Drosophila, maternal morphogens control the patterned activation of gap genes, which encode transcriptional regulators that shape the patterned expression of pair-rule genes. This patterning cascade takes place before cellularization. Pair-rule gene products subsequently `imprint' segment polarity genes with reiterated patterns, thus defining the primordial segments. This mechanism must be greatly modified in insect groups in which many segments emerge only after cellularization. In beetles and parasitic wasps, for instance, pair-rule homologues are expressed in patterns consistent with roles during segmentation, but these patterns emerge within cellular fields. In contrast, although in locusts pair-rule homologues may not control segmentation, some segment polarity genes and their interactions are conserved. Perhaps segmentation is modular, with each module autonomously expressing a characteristic intrinsic behaviour in response to transient stimuli. If so, evolution could rearrange inputs to modules without changing their intrinsic behaviours. Here we suggest, using computer simulations, that the Drosophila segment polarity genes constitute such a module, and that this module is resistant to variations in the kinetic constants that govern its behaviour.

Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks.

PubMed

Wu, Siqi; Joseph, Antony; Hammonds, Ann S; Celniker, Susan E; Yu, Bin; Frise, Erwin

2016-04-19

Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set ofDrosophilaearly embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identified 21 principal patterns (PP). Providing a compact yet biologically interpretable representation ofDrosophilaexpression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. The performance of PP with theDrosophiladata suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.

Capsaicin-induced activation of ERK1/2 and its involvement in GAP-43 expression and CGRP depletion in organotypically cultured DRG neurons.

PubMed

Li, Yunfeng; Liu, Guixiang; Li, Hao; Xu, Youzheng; Zhang, Hong; Liu, Zhen

2013-04-01

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.

Gene expression of osteogenic factors following gene therapy in mandibular lengthening.

PubMed

Wu, Guoping; Zhou, Bin; Hu, Chunbing; Li, Shaolan

2015-03-01

This study investigated the effect of gene therapy on the expression of osteogenic mediators in mandibular distraction osteogenesis rabbits. Bilateral mandibular osteotomies were performed in 45 New-Zealand rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/d for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups: 2 μg (0.1 μg/μL) of recombinant plasmid pIRES-hVEGF165-hBMP-2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES, and the same volume of normal saline were injected into the distraction gap of groups A, B, C, D, and E, respectively, followed by electroporation. Three animals were killed at the 7th, 14th, and 28th day after gene transfected in different groups, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations; the mean optic densities (MODs) and integral optical density of bone morphogenetic protein (BMP-2) and transforming growth factor β1 (TGF-β1)-positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with SPSS (SPSS Inc, Chicago, IL). Bone morphogenetic protein 2 and TGF-β1 staining was mainly located in inflammatory cells, monocytes, fibroblasts, osteoblasts, osteocytes, and chondrocytes in the distraction zones. Their strongest expression reached to the peak at the seventh day and decreased at the 14th day of consolidation stage; at the 28th day, they expressed weakly. Image analysis results show that, at the seventh day, the expression of BMP-2 in group B (0.26 ± 0.03, 0.36 ± 0.02) was the strongest; there was significant difference among them (P < 0.01), whereas the expression of TGF-β1 in group C (0.38 ± 0.06, 1.05 ± 0.19) is strongest followed by group A (0.34 ± 0.05, 0.95 ± 0.16) and B (0.33 ± 0.07, 0.90 ± 0.19). At every time point, the level of expression of BMP-2 and TGF-β1 in gene therapy groups (groups A, B, and C) was remarkably higher than those in non-gene therapy groups(groups D and E). There were significant differences between gene therapy groups and non-gene therapy groups (P < 0.05 or P < 0.001). These results indicated that local gene transfection can up-regulate the expression of osteogenic mediators (BMP-2 and TGF-β1), which may promote cell differentiation and proliferation and stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Siqi; Joseph, Antony; Hammonds, Ann S.

Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set of Drosophila early embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identifiedmore » 21 principal patterns (PP). Providing a compact yet biologically interpretable representation of Drosophila expression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. In conclusion, the performance of PP with the Drosophila data suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.« less

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

PubMed Central

Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

2014-01-01

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

Stability-driven nonnegative matrix factorization to interpret spatial gene expression and build local gene networks

DOE PAGES

Wu, Siqi; Joseph, Antony; Hammonds, Ann S.; ...

2016-04-06

Spatial gene expression patterns enable the detection of local covariability and are extremely useful for identifying local gene interactions during normal development. The abundance of spatial expression data in recent years has led to the modeling and analysis of regulatory networks. The inherent complexity of such data makes it a challenge to extract biological information. We developed staNMF, a method that combines a scalable implementation of nonnegative matrix factorization (NMF) with a new stability-driven model selection criterion. When applied to a set of Drosophila early embryonic spatial gene expression images, one of the largest datasets of its kind, staNMF identifiedmore » 21 principal patterns (PP). Providing a compact yet biologically interpretable representation of Drosophila expression patterns, PP are comparable to a fate map generated experimentally by laser ablation and show exceptional promise as a data-driven alternative to manual annotations. Our analysis mapped genes to cell-fate programs and assigned putative biological roles to uncharacterized genes. Finally, we used the PP to generate local transcription factor regulatory networks. Spatially local correlation networks were constructed for six PP that span along the embryonic anterior-posterior axis. Using a two-tail 5% cutoff on correlation, we reproduced 10 of the 11 links in the well-studied gap gene network. In conclusion, the performance of PP with the Drosophila data suggests that staNMF provides informative decompositions and constitutes a useful computational lens through which to extract biological insight from complex and often noisy gene expression data.« less

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

PubMed Central

Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

2013-01-01

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy. PMID:23861780

Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation.

PubMed

Tsai, Sen-Wei; Tung, Yu-Tang; Chen, Hsiao-Ling; Yang, Shang-Hsun; Liu, Chia-Yi; Lu, Michelle; Pai, Hui-Jing; Lin, Chi-Chen; Chen, Chuan-Mu

2016-02-01

Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (p<0.05). And enhancement of the gene expression of the muscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

Computational Analysis and Low-Scale Constitutive Expression of Laccases Synthetic Genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris

PubMed Central

Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A.; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M.

2015-01-01

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications. PMID:25611746

Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

PubMed

Rivera-Hoyos, Claudia M; Morales-Álvarez, Edwin David; Poveda-Cuevas, Sergio Alejandro; Reyes-Guzmán, Edwin Alfredo; Poutou-Piñales, Raúl A; Reyes-Montaño, Edgar Antonio; Pedroza-Rodríguez, Aura Marina; Rodríguez-Vázquez, Refugio; Cardozo-Bernal, Ángela M

2015-01-01

Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range of substrates that laccases can transform. This contributes to a great gamut of products in diverse settings: industry, clinical and chemical use, and environmental applications.

How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation

PubMed Central

Schroeder, Mark D.; Greer, Christina; Gaul, Ulrike

2011-01-01

The generation of metameric body plans is a key process in development. In Drosophila segmentation, periodicity is established rapidly through the complex transcriptional regulation of the pair-rule genes. The ‘primary’ pair-rule genes generate their 7-stripe expression through stripe-specific cis-regulatory elements controlled by the preceding non-periodic maternal and gap gene patterns, whereas ‘secondary’ pair-rule genes are thought to rely on 7-stripe elements that read off the already periodic primary pair-rule patterns. Using a combination of computational and experimental approaches, we have conducted a comprehensive systems-level examination of the regulatory architecture underlying pair-rule stripe formation. We find that runt (run), fushi tarazu (ftz) and odd skipped (odd) establish most of their pattern through stripe-specific elements, arguing for a reclassification of ftz and odd as primary pair-rule genes. In the case of run, we observe long-range cis-regulation across multiple intervening genes. The 7-stripe elements of run, ftz and odd are active concurrently with the stripe-specific elements, indicating that maternal/gap-mediated control and pair-rule gene cross-regulation are closely integrated. Stripe-specific elements fall into three distinct classes based on their principal repressive gap factor input; stripe positions along the gap gradients correlate with the strength of predicted input. The prevalence of cis-elements that generate two stripes and their genomic organization suggest that single-stripe elements arose by splitting and subfunctionalization of ancestral dual-stripe elements. Overall, our study provides a greatly improved understanding of how periodic patterns are established in the Drosophila embryo. PMID:21693522

Dual Analysis of the Murine Cytomegalovirus and Host Cell Transcriptomes Reveal New Aspects of the Virus-Host Cell Interface

PubMed Central

Juranic Lisnic, Vanda; Babic Cac, Marina; Lisnic, Berislav; Trsan, Tihana; Mefferd, Adam; Das Mukhopadhyay, Chitrangada; Cook, Charles H.; Jonjic, Stipan; Trgovcich, Joanne

2013-01-01

Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. PMID:24086132

Coordinated Gene Expression of Neuroinflammatory and Cell Signaling Markers in Dorsolateral Prefrontal Cortex during Human Brain Development and Aging

PubMed Central

Primiani, Christopher T.; Ryan, Veronica H.; Rao, Jagadeesh S.; Cam, Margaret C.; Ahn, Kwangmi; Modi, Hiren R.; Rapoport, Stanley I.

2014-01-01

Background Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. Hypothesis Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. Methods We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. Results Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. Conclusions Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate throughout the lifespan underlie different phenotypic processes during Aging compared to Development. PMID:25329999

Coordinated gene expression of neuroinflammatory and cell signaling markers in dorsolateral prefrontal cortex during human brain development and aging.

PubMed

Primiani, Christopher T; Ryan, Veronica H; Rao, Jagadeesh S; Cam, Margaret C; Ahn, Kwangmi; Modi, Hiren R; Rapoport, Stanley I

2014-01-01

Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate throughout the lifespan underlie different phenotypic processes during Aging compared to Development.

Expression of Immediate-Early Genes in the Inferior Colliculus and Auditory Cortex in Salicylate-Induced Tinnitus in Rat

PubMed Central

Hu, S.S.; Mei, L.; Chen, J.Y.; Huang, Z.W.; Wu, H.

2014-01-01

Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG) expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc) and the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS) paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC) and auditory cortex (AC), in contradiction of our hypothesis. Expression of N-methyl D-aspartate receptor subunit 2B (NR2B) was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus. PMID:24704997

Cell-Free Optogenetic Gene Expression System.

PubMed

Jayaraman, Premkumar; Yeoh, Jing Wui; Jayaraman, Sudhaghar; Teh, Ai Ying; Zhang, Jingyun; Poh, Chueh Loo

2018-04-20

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

Combining mouse mammary gland gene expression and comparative mapping for the identification of candidate genes for QTL of milk production traits in cattle

PubMed Central

Ron, Micha; Israeli, Galit; Seroussi, Eyal; Weller, Joel I; Gregg, Jeffrey P; Shani, Moshe; Medrano, Juan F

2007-01-01

Background Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination. Results We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas. Conclusion This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cgQTL can be used to determine priority of candidate genes for QTN analysis based on differential expression in the target organ. PMID:17584498

Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.

PubMed

Haag, Daniel; Zipper, Petra; Westrich, Viola; Karra, Daniela; Pfleger, Karin; Toedt, Grischa; Blond, Frederik; Delhomme, Nicolas; Hahn, Meinhard; Reifenberger, Julia; Reifenberger, Guido; Lichter, Peter

2012-01-01

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.

Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis.

PubMed

El-Nour, H; Lundeberg, L; Al-Tawil, R; Granlund, A; Lonne-Rahm, S-B; Nordlind, K

2006-01-01

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.

Supervised group Lasso with applications to microarray data analysis

PubMed Central

Ma, Shuangge; Song, Xiao; Huang, Jian

2007-01-01

Background A tremendous amount of efforts have been devoted to identifying genes for diagnosis and prognosis of diseases using microarray gene expression data. It has been demonstrated that gene expression data have cluster structure, where the clusters consist of co-regulated genes which tend to have coordinated functions. However, most available statistical methods for gene selection do not take into consideration the cluster structure. Results We propose a supervised group Lasso approach that takes into account the cluster structure in gene expression data for gene selection and predictive model building. For gene expression data without biological cluster information, we first divide genes into clusters using the K-means approach and determine the optimal number of clusters using the Gap method. The supervised group Lasso consists of two steps. In the first step, we identify important genes within each cluster using the Lasso method. In the second step, we select important clusters using the group Lasso. Tuning parameters are determined using V-fold cross validation at both steps to allow for further flexibility. Prediction performance is evaluated using leave-one-out cross validation. We apply the proposed method to disease classification and survival analysis with microarray data. Conclusion We analyze four microarray data sets using the proposed approach: two cancer data sets with binary cancer occurrence as outcomes and two lymphoma data sets with survival outcomes. The results show that the proposed approach is capable of identifying a small number of influential gene clusters and important genes within those clusters, and has better prediction performance than existing methods. PMID:17316436

Cytotoxic effect of the Her-2/Her-1 inhibitor PKI-166 on renal cancer cells expressing the connexin 32 gene.

PubMed

Fujimoto, Eriko; Yano, Tomohiro; Sato, Hiromi; Hagiwara, Kiyokazu; Yamasaki, Hiroshi; Shirai, Sumiko; Fukumoto, Keiko; Hagiwara, Hiromi; Negishi, Etsuko; Ueno, Koichi

2005-02-01

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis.

Connexin 30 expression inhibits growth of human malignant gliomas but protects them against radiation therapy

PubMed Central

Artesi, Maria; Kroonen, Jerome; Bredel, Markus; Nguyen-Khac, Minh; Deprez, Manuel; Schoysman, Laurent; Poulet, Christophe; Chakravarti, Arnab; Kim, Hyunsoo; Scholtens, Denise; Seute, Tatjana; Rogister, Bernard; Bours, Vincent; Robe, Pierre A.

2015-01-01

Background Glioblastomas remain ominous tumors that almost invariably escape treatment. Connexins are a family of transmembrane, gap junction-forming proteins, some members of which were reported to act as tumor suppressors and to modulate cellular metabolism in response to cytotoxic stress. Methods We analyzed the copy number and expression of the connexin (Cx)30 gene gap junction beta-6 (GJB6), as well as of its protein immunoreactivity in several public and proprietary repositories of glioblastomas, and their influence on patient survival. We evaluated the effect of the expression of this gap junction protein on the growth, DNA repair and energy metabolism, and treatment resistance of these tumors. Results The GJB6 gene was deleted in 25.8% of 751 analyzed tumors and mutated in 15.8% of 158 tumors. Cx30 immunoreactivity was absent in 28.9% of 145 tumors. Restoration of Cx30 expression in human glioblastoma cells reduced their growth in vitro and as xenografts in the striatum of immunodeficient mice. Cx30 immunoreactivity was, however, found to adversely affect survival in 2 independent retrospective cohorts of glioblastoma patients. Cx30 was found in clonogenic assays to protect glioblastoma cells against radiation-induced mortality and to decrease radiation-induced DNA damage. This radioprotection correlated with a heat shock protein 90–dependent mitochondrial translocation of Cx30 following radiation and an improved ATP production following this genotoxic stress. Conclusion These results underline the complex relationship between potential tumor suppressors and treatment resistance in glioblastomas and single out GJB6/Cx30 as a potential biomarker and target for therapeutic intervention in these tumors. PMID:25155356

A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death

PubMed Central

Minogue, Peter J.; Tong, Jun-Jie; Arora, Anita; Russell-Eggitt, Isabelle; Hunt, David M.; Moore, Anthony T.; Ebihara, Lisa; Beyer, Eric C.; Berthoud, Viviana M.

2009-01-01

PURPOSE To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. METHODS The GJA8 gene was directly sequenced. Formation of functional channels was assessed by two-microelectrode voltage-clamp. Connexin protein levels and distribution were assessed by immunoblotting and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. RESULTS Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in cell number and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. CONCLUSIONS Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function. PMID:19684000

Speed regulation of genetic cascades allows for evolvability in the body plan specification of insects

PubMed Central

Zhu, Xin; Rudolf, Heike; Healey, Lucas; François, Paul; Brown, Susan J.; Klingler, Martin; El-Sherif, Ezzat

2017-01-01

During the anterior−posterior fate specification of insects, anterior fates arise in a nonelongating tissue (called the “blastoderm”), and posterior fates arise in an elongating tissue (called the “germband”). However, insects differ widely in the extent to which anterior−posterior fates are specified in the blastoderm versus the germband. Here we present a model in which patterning in both the blastoderm and germband of the beetle Tribolium castaneum is based on the same flexible mechanism: a gradient that modulates the speed of a genetic cascade of gap genes, resulting in the induction of sequential kinematic waves of gap gene expression. The mechanism is flexible and capable of patterning both elongating and nonelongating tissues, and hence converting blastodermal to germband fates and vice versa. Using RNAi perturbations, we found that blastodermal fates could be shifted to the germband, and germband fates could be generated in a blastoderm-like morphology. We also suggest a molecular mechanism underlying our model, in which gradient levels regulate the switch between two enhancers: One enhancer is responsible for sequential gene activation, and the other is responsible for freezing temporal rhythms into spatial patterns. This model is consistent with findings in Drosophila melanogaster, where gap genes were found to be regulated by two nonredundant “shadow” enhancers. PMID:28973882

Gene expression networks underlying ovarian development in wild largemouth bass (Micropterus salmoides).

PubMed

Martyniuk, Christopher J; Prucha, Melinda S; Doperalski, Nicholas J; Antczak, Philipp; Kroll, Kevin J; Falciani, Francesco; Barber, David S; Denslow, Nancy D

2013-01-01

Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation.

Gap junctions are essential for murine primordial follicle assembly immediately before birth.

PubMed

Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua

2016-02-01

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking. © 2016 Society for Reproduction and Fertility.

Aberrant expression of long noncoding RNAs in autistic brain.

PubMed

Ziats, Mark N; Rennert, Owen M

2013-03-01

The autism spectrum disorders (ASD) have a significant hereditary component, but the implicated genetic loci are heterogeneous and complex. Consequently, there is a gap in understanding how diverse genomic aberrations all result in one clinical ASD phenotype. Gene expression studies from autism brain tissue have demonstrated that aberrantly expressed protein-coding genes may converge onto common molecular pathways, potentially reconciling the strong heritability and shared clinical phenotypes with the genomic heterogeneity of the disorder. However, the regulation of gene expression is extremely complex and governed by many mechanisms, including noncoding RNAs. Yet no study in ASD brain tissue has assessed for changes in regulatory long noncoding RNAs (lncRNAs), which represent a large proportion of the human transcriptome, and actively modulate mRNA expression. To assess if aberrant expression of lncRNAs may play a role in the molecular pathogenesis of ASD, we profiled over 33,000 annotated lncRNAs and 30,000 mRNA transcripts from postmortem brain tissue of autistic and control prefrontal cortex and cerebellum by microarray. We detected over 200 differentially expressed lncRNAs in ASD, which were enriched for genomic regions containing genes related to neurodevelopment and psychiatric disease. Additionally, comparison of differences in expression of mRNAs between prefrontal cortex and cerebellum within individual donors showed ASD brains had more transcriptional homogeneity. Moreover, this was also true of the lncRNA transcriptome. Our results suggest that further investigation of lncRNA expression in autistic brain may further elucidate the molecular pathogenesis of this disorder.

Cortical Auditory Deafferentation Induces Long-Term Plasticity in the Inferior Colliculus of Adult Rats: Microarray and qPCR Analysis

PubMed Central

Clarkson, Cheryl; Herrero-Turrión, M. Javier; Merchán, Miguel A.

2012-01-01

The cortico-collicular pathway is a bilateral excitatory projection from the cortex to the inferior colliculus (IC). It is asymmetric and predominantly ipsilateral. Using microarrays and RT-qPCR we analyzed changes in gene expression in the IC after unilateral lesions of the auditory cortex, comparing the ICs ipsi- and contralateral to the lesioned side. At 15 days after surgery there were mainly changes in gene expression in the IC ipsilateral to the lesion. Regulation primarily involved inflammatory cascade genes, suggesting a direct effect of degeneration rather than a neuronal plastic reorganization. Ninety days after the cortical lesion the ipsilateral IC showed a significant up-regulation of genes involved in apoptosis and axonal regeneration combined with a down-regulation of genes involved in neurotransmission, synaptic growth, and gap junction assembly. In contrast, the contralateral IC at 90 days post-lesion showed an up-regulation in genes primarily related to neurotransmission, cell proliferation, and synaptic growth. There was also a down-regulation in autophagy and neuroprotection genes. These findings suggest that the reorganization in the IC after descending pathway deafferentation is a long-term process involving extensive changes in gene expression regulation. Regulated genes are involved in many different neuronal functions, and the number and gene rearrangement profile seems to depend on the density of loss of the auditory cortical inputs. PMID:23233834

Hopkins works with the Vaccine-21 GAP

NASA Image and Video Library

2014-01-15

ISS038-E-031400 (14 Jan. 2014) --- NASA astronaut Mike Hopkins, Expedition 38 flight engineer, accesses the Commercial Generic Bioprocessing Apparatus-2 (CGBA-2) while working with the Vaccine-21 Group Activation Pack (GAP) experiment in the Harmony node of the International Space Station. This experiment also referred to as Antibiotic Effectiveness in Space-1 (AES-1) tests the hypothesis that antibiotics used to treat bacterial grown in space will exhibit reduced efficacy and will be associated with specific changes in bacterial gene expression that correlate with cell survival.

Mastracchio works with the Vaccine-21 GAP

NASA Image and Video Library

2014-01-16

ISS038-E-031407 (16 Jan. 2014) --- NASA astronaut Rick Mastracchio, Expedition 38 flight engineer, accesses the Commercial Generic Bioprocessing Apparatus-2 (CGBA-2) while working with the Vaccine-21 Group Activation Pack (GAP) experiment in the Harmony node of the International Space Station. This experiment also referred to as Antibiotic Effectiveness in Space-1 (AES-1) tests the hypothesis that antibiotics used to treat bacterial grown in space will exhibit reduced efficacy and will be associated with specific changes in bacterial gene expression that correlate with cell survival.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimelman, Aya; Levy, Asaf; Sberro, Hila

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes our assays revealed novel toxins and restriction enzymes, and new classes of smallmore » non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator dnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.« less

Leaps and lulls in the developmental transcriptome of Dictyostelium discoideum.

PubMed

Rosengarten, Rafael David; Santhanam, Balaji; Fuller, Danny; Katoh-Kurasawa, Mariko; Loomis, William F; Zupan, Blaz; Shaulsky, Gad

2015-04-13

Development of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under both developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods. Here, we combine the superior depth and specificity of RNA-seq-based analysis of mRNA abundance with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large shifts. For each time point we treated the entire transcriptome as single phenotype, and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represented gradual changes in mRNA abundance, or molecular phenotype, and the gaps represented times during which many genes are differentially expressed rapidly, and thus the phenotype changes dramatically. Comparing developmental experiments revealed that gene expression in filter developed cells lagged behind those treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis suggested that the transition to multicellularity coincided with rapid accumulation of transcripts associated with DNA processes and mitosis. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. Our analysis also demonstrated a high level of synchrony among the developing structures throughout development. Our data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and among multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for studies of developmental gene expression.

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

PubMed

Santana, Flávia A; Nunes, Francis M F; Vieira, Carlos U; Machado, Maria Alice M S; Kerr, Warwick E; Silva, Wilson A; Bonetti, Ana Maria

2006-03-01

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

Bridging the Gap between Gene Expression and Metabolic Phenotype via Kinetic Models

DTIC Science & Technology

2013-07-22

construction of large-scale kinetic models of metabolism, namely, the detailed definition of appro- priate reaction rate expressions and the determination...mole of bio- mass precursor, and the summation included only the drain fluxes to biomass. Note that this definition of the biomass growth rate can... 4P G ly co ly si s Glu Pro Arg Lys Glucose-6P Carbohydrates RNA Lipids Acetaldehyde Figure 2 Metabolic network of the central carbon

Strand-specific RNA-seq analysis of the Lactobacillus delbrueckii subsp. bulgaricus transcriptome.

PubMed

Zheng, Huajun; Liu, Enuo; Shi, Tao; Ye, Luyi; Konno, Tomonobu; Oda, Munehiro; Ji, Zai-Si

2016-02-01

Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lb. bulgaricus 2038) is an industrial bacterium that is used as a starter for dairy products. We proposed several hypotheses concerning its industrial features previously. Here, we utilized RNA-seq to explore the transcriptome of Lb. bulgaricus 2038 from four different growth phases under whey conditions. The most abundantly expressed genes in the four stages were mainly involved in translation (for the logarithmic stage), glycolysis (for control/lag stages), lactic acid production (all the four stages), and 10-formyl tetrahydrofolate production (for the stationary stage). The high expression of genes like d-lactate dehydrogenase was thought as a result of energy production, and consistent expression of EPS synthesis genes, the restriction-modification (RM) system and the CRISPR/Cas system were validated for explaining the advantage of this strain in yoghurt production. Several postulations, like NADPH production through GapN bypass, converting aspartate into carbon-skeleton intermediates, and formate production through degrading GTP, were proved not working under these culture conditions. The high expression of helicase genes and co-expressed amino acids/oligopeptides transporting proteins indicated that the helicase might mediate the strain obtaining nitrogen source from the environment. The transport system of Lb. bulgaricus 2038 was found to be regulated by antisense RNA, hinting the potential application of non-coding RNA in regulating lactic acid bacteria (LAB) gene expression. Our study has primarily uncovered Lb. bulgaricus 2038 transcriptome, which could gain a better understanding of the regulation system in Lb. bulgaricus and promote its industrial application.

Expression and Function of Connexin 43 in Human Gingival Wound Healing and Fibroblasts

PubMed Central

Tarzemany, Rana; Jiang, Guoqiao; Larjava, Hannu; Häkkinen, Lari

2015-01-01

Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva. PMID:25584940

Dual mode of embryonic development is highlighted by expression and function of Nasonia pair-rule genes

PubMed Central

Rosenberg, Miriam I; Brent, Ava E; Payre, François; Desplan, Claude

2014-01-01

Embryonic anterior–posterior patterning is well understood in Drosophila, which uses ‘long germ’ embryogenesis, in which all segments are patterned before cellularization. In contrast, most insects use ‘short germ’ embryogenesis, wherein only head and thorax are patterned in a syncytial environment while the remainder of the embryo is generated after cellularization. We use the wasp Nasonia (Nv) to address how the transition from short to long germ embryogenesis occurred. Maternal and gap gene expression in Nasonia suggest long germ embryogenesis. However, the Nasonia pair-rule genes even-skipped, odd-skipped, runt and hairy are all expressed as early blastoderm pair-rule stripes and late-forming posterior stripes. Knockdown of Nv eve, odd or h causes loss of alternate segments at the anterior and complete loss of abdominal segments. We propose that Nasonia uses a mixed mode of segmentation wherein pair-rule genes pattern the embryo in a manner resembling Drosophila at the anterior and ancestral Tribolium at the posterior. DOI: http://dx.doi.org/10.7554/eLife.01440.001 PMID:24599282

BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration

NASA Astrophysics Data System (ADS)

Gao, Mingyong; Lu, Paul; Lynam, Dan; Bednark, Bridget; Campana, W. Marie; Sakamoto, Jeff; Tuszynski, Mark

2016-12-01

Objective. We combined implantation of multi-channel templated agarose scaffolds with growth factor gene delivery to examine whether this combinatorial treatment can enhance peripheral axonal regeneration through long sciatic nerve gaps. Approach. 15 mm long scaffolds were templated into highly organized, strictly linear channels, mimicking the linear organization of natural nerves into fascicles of related function. Scaffolds were filled with syngeneic bone marrow stromal cells (MSCs) secreting the growth factor brain derived neurotrophic factor (BDNF), and lentiviral vectors expressing BDNF were injected into the sciatic nerve segment distal to the scaffold implantation site. Main results. Twelve weeks after injury, scaffolds supported highly linear regeneration of host axons across the 15 mm lesion gap. The incorporation of BDNF-secreting cells into scaffolds significantly increased axonal regeneration, and additional injection of viral vectors expressing BDNF into the distal segment of the transected nerve significantly enhanced axonal regeneration beyond the lesion. Significance. Combinatorial treatment with multichannel bioengineered scaffolds and distal growth factor delivery significantly improves peripheral nerve repair, rivaling the gold standard of autografts.

Notch3 is necessary for blood vessel integrity in the central nervous system.

PubMed

Henshall, Tanya L; Keller, Annika; He, Liqun; Johansson, Bengt R; Wallgard, Elisabet; Raschperger, Elisabeth; Mäe, Maarja Andaloussi; Jin, Shaobo; Betsholtz, Christer; Lendahl, Urban

2015-02-01

Vascular smooth muscle cells (VSMC) are important for contraction, blood flow distribution, and regulation of blood vessel diameter, but to what extent they contribute to the integrity of blood vessels and blood-brain barrier function is less well understood. In this report, we explored the impact of the loss of VSMC in the Notch3(-/-) mouse on blood vessel integrity in the central nervous system. Notch3(-/-) mice showed focal disruptions of the blood-brain barrier demonstrated by extravasation of tracers accompanied by fibrin deposition in the retinal vasculature. This blood-brain barrier leakage was accompanied by a regionalized and patchy loss of VSMC, with VSMC gaps predominantly in arterial resistance vessels of larger caliber. The loss of VSMC appeared to be caused by progressive degeneration of VSMC resulting in a gradual loss of VSMC marker expression and a progressive acquisition of an aberrant VSMC phenotype closer to the gaps, followed by enhanced apoptosis and cellular disintegration in the gaps. Arterial VSMC were the only mural cell type that was morphologically affected, despite Notch3 also being expressed in pericytes. Transcriptome analysis of isolated brain microvessels revealed gene expression changes in Notch3(-/-) mice consistent with loss of arterial VSMC and presumably secondary transcriptional changes were observed in endothelial genes, which may explain the compromised vascular integrity. We demonstrate that Notch3 is important for survival of VSMC, and reveal a critical role for Notch3 and VSMC in blood vessel integrity and blood-brain barrier function in the mammalian vasculature. © 2014 American Heart Association, Inc.

Evolution and Expression of Tissue Globins in Ray-Finned Fishes.

PubMed

Gallagher, Michael D; Macqueen, Daniel J

2017-01-01

The globin gene family encodes oxygen-binding hemeproteins conserved across the major branches of multicellular life. The origins and evolutionary histories of complete globin repertoires have been established for many vertebrates, but there remain major knowledge gaps for ray-finned fish. Therefore, we used phylogenetic, comparative genomic and gene expression analyses to discover and characterize canonical “non-blood” globin family members (i.e., myoglobin, cytoglobin, neuroglobin, globin-X, and globin-Y) across multiple ray-finned fish lineages, revealing novel gene duplicates (paralogs) conserved from whole genome duplication (WGD) and small-scale duplication events. Our key findings were that: (1) globin-X paralogs in teleosts have been retained from the teleost-specific WGD, (2) functional paralogs of cytoglobin, neuroglobin, and globin-X, but not myoglobin, have been conserved from the salmonid-specific WGD, (3) triplicate lineage-specific myoglobin paralogs are conserved in arowanas (Osteoglossiformes), which arose by tandem duplication and diverged under positive selection, (4) globin-Y is retained in multiple early branching fish lineages that diverged before teleosts, and (5) marked variation in tissue-specific expression of globin gene repertoires exists across ray-finned fish evolution, including several previously uncharacterized sites of expression. In this respect, our data provide an interesting link between myoglobin expression and the evolution of air breathing in teleosts. Together, our findings demonstrate great-unrecognized diversity in the repertoire and expression of nonblood globins that has arisen during ray-finned fish evolution.

Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

NASA Astrophysics Data System (ADS)

Bosak, T.; Pearson, A.; Losick, R.

2005-12-01

Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

PubMed Central

Zermiani, Monica; Zonin, Elisabetta; Nonis, Alberto; Begheldo, Maura; Ceccato, Luca; Vezzaro, Alice; Baldan, Barbara; Trentin, Annarita; Masi, Antonio; Pegoraro, Marco; Fadanelli, Livio; Teale, William; Palme, Klaus; Quintieri, Luigi; Ruperti, Benedetto

2015-01-01

Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. ‘Superficial scald’ of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress. PMID:26428066

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes.

PubMed

Zhong, Xingyu; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

2013-07-01

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

Coregulation of srGAP1 by Wnt and Androgen Receptor Signaling: A New Target for Treatment of CRPC

DTIC Science & Technology

2014-10-01

hormone naïve prostate cancer. This direct interaction between AR and β-catenin seemed to elicit a specific expression of a set of target genes in low...nohistochemistry staining in 212 prostate can- cer specimens, including 122 localized pros - tate cancer from prostectomy specimens and 90 from CRPC specimens from...significant down-regulation of membra- nous β-catenin expression in prostate cancer compared to benign prostatic glands and an associatation with

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

PubMed

Dey-Rao, Rama; Sinha, Animesh A

2017-01-28

Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address a major gap in knowledge regarding the systemic changes underlying skin-specific manifestation of vitiligo. Several transcriptional "hot spots" observed in both environments offer prioritized targets for identifying disease risk genes. Finally, within the transcriptional framework of VL, we identify five novel molecules (STAT1, PRKCD, PTPN6, MYC and FGFR2) that lend themselves to being targeted by drugs for future potential VL-therapy.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

Genetic basis of priority effects: insights from nectar yeast

PubMed Central

Hartwig, Thomas

2016-01-01

Priority effects, in which the order of species arrival dictates community assembly, can have a major influence on species diversity, but the genetic basis of priority effects remains unknown. Here, we suggest that nitrogen scavenging genes previously considered responsible for starvation avoidance may drive priority effects by causing rapid resource depletion. Using single-molecule sequencing, we de novo assembled the genome of the nectar-colonizing yeast, Metschnikowia reukaufii, across eight scaffolds and complete mitochondrion, with gap-free coverage over gene spaces. We found a high rate of tandem gene duplication in this genome, enriched for nitrogen metabolism and transport. Both high-capacity amino acid importers, GAP1 and PUT4, present as tandem gene arrays, were highly expressed in synthetic nectar and regulated by the availability and quality of amino acids. In experiments with competitive nectar yeast, Candida rancensis, amino acid addition alleviated suppression of C. rancensis by early arrival of M. reukaufii, corroborating that amino acid scavenging may contribute to priority effects. Because niche pre-emption via rapid resource depletion may underlie priority effects in a broad range of microbial, plant and animal communities, nutrient scavenging genes like the ones we considered here may be broadly relevant to understanding priority effects. PMID:27708148

Knockdown of connexin43-mediated regulation of the zone of polarizing activity in the developing chick limb leads to digit truncation.

PubMed

Law, Lee Yong; Lin, Jun Sheng; Becker, David L; Green, Colin R

2002-12-01

In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.

Gene expression analysis in MCF-7 breast cancer cells treated with recombinant bromelain.

PubMed

Fouz, Nour; Amid, Azura; Hashim, Yumi Zuhanis Has-Yun

2014-08-01

The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach.

PubMed

Ben Azoun, Safa; Kallel, Héla

2017-08-01

Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

RedeR: R/Bioconductor package for representing modular structures, nested networks and multiple levels of hierarchical associations

PubMed Central

2012-01-01

Visualization and analysis of molecular networks are both central to systems biology. However, there still exists a large technological gap between them, especially when assessing multiple network levels or hierarchies. Here we present RedeR, an R/Bioconductor package combined with a Java core engine for representing modular networks. The functionality of RedeR is demonstrated in two different scenarios: hierarchical and modular organization in gene co-expression networks and nested structures in time-course gene expression subnetworks. Our results demonstrate RedeR as a new framework to deal with the multiple network levels that are inherent to complex biological systems. RedeR is available from http://bioconductor.org/packages/release/bioc/html/RedeR.html. PMID:22531049

Immunological profile in a family with nephrogenic diabetes insipidus with a novel 11 kb deletion in AVPR2 and ARHGAP4 genes

PubMed Central

Fujimoto, Masaya; Imai, Kohsuke; Hirata, Kenji; Kashiwagi, Reiichi; Morinishi, Yoichi; Kitazawa, Katsuhiko; Sasaki, Sei; Arinami, Tadao; Nonoyama, Shigeaki; Noguchi, Emiko

2008-01-01

Background Congenital nephrogenic diabetes insipidus (NDI) is characterised by an inability to concentrate urine despite normal or elevated plasma levels of the antidiuretic hormone arginine vasopressin. We report a Japanese extended family with NDI caused by an 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the Rho GTPase-activating protein 4 (ARHGAP4) locus. ARHGAP4 belongs to the RhoGAP family, Rho GTPases are critical regulators of many cellular activities, such as motility and proliferation which enhances intrinsic GTPase activity. ARHGAP4 is expressed at high levels in hematopoietic cells, and it has been reported that an NDI patient lacking AVPR2 and all of ARHGAP4 showed immunodeficiency characterised by a marked reduction in the number of circulating CD3+ cells and almost complete absence of CD8+ cells. Methods PCR and sequencing were performed to identify the deleted region in the Japanese NDI patients. Immunological profiles of the NDI patients were analysed by flow cytometry. We also investigated the gene expression profiles of peripheral blood mononuclear cells (PBMC) from NDI patients and healthy controls in microarray technique. Results We evaluated subjects (one child and two adults) with 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the ARHGAP4. Hematologic tests showed a reduction of CD4+ cells in one adult patient, a reduction in CD8+ cells in the paediatric patient, and a slight reduction in the serum IgG levels in the adult patients, but none of them showed susceptibility to infection. Gene expression profiling of PBMC lacking ARHGAP4 revealed that expression of RhoGAP family genes was not influenced greatly by the lack of ARHGAP4. Conclusion These results suggest that loss of ARHGAP4 expression is not compensated for by other family members. ARHGAP4 may play some role in lymphocyte differentiation but partial loss of ARHGAP4 does not result in clinical immunodeficiency. PMID:18489790

Metabolic engineering for improved production of ethanol by Corynebacterium glutamicum.

PubMed

Jojima, Toru; Noburyu, Ryoji; Sasaki, Miho; Tajima, Takahisa; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

2015-02-01

Recombinant Corynebacterium glutamicum harboring genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) can produce ethanol under oxygen deprivation. We investigated the effects of elevating the expression levels of glycolytic genes, as well as pdc and adhB, on ethanol production. Overexpression of four glycolytic genes (pgi, pfkA, gapA, and pyk) in C. glutamicum significantly increased the rate of ethanol production. Overexpression of tpi, encoding triosephosphate isomerase, further enhanced productivity. Elevated expression of pdc and adhB increased ethanol yield, but not the rate of production. Fed-batch fermentation using an optimized strain resulted in ethanol production of 119 g/L from 245 g/L glucose with a yield of 95% of the theoretical maximum. Further metabolic engineering, including integration of the genes for xylose and arabinose metabolism, enabled consumption of glucose, xylose, and arabinose, and ethanol production (83 g/L) at a yield of 90 %. This study demonstrated that C. glutamicum has significant potential for the production of cellulosic ethanol.

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen

PubMed Central

Airoldi, Edoardo M.; Miller, Darach; Athanasiadou, Rodoniki; Brandt, Nathan; Abdul-Rahman, Farah; Neymotin, Benjamin; Hashimoto, Tatsu; Bahmani, Tayebeh; Gresham, David

2016-01-01

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. In Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate–controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source–specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genes GAP1, MEP2, DAL5, PUT4, and DIP5. Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments. PMID:26941329

Ras-Driven Transcriptome Analysis Identifies Aurora Kinase A as a Potential Malignant Peripheral Nerve Sheath Tumor Therapeutic Target

PubMed Central

Patel, Ami V.; Eaves, David; Jessen, Walter J.; Rizvi, Tilat A.; Ecsedy, Jeffrey A.; Qian, Mark G.; Aronow, Bruce J.; Perentesis, John P.; Serra, Eduard; Cripe, Timothy P.; Miller, Shyra J.; Ratner, Nancy

2013-01-01

Purpose Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase. Results We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. PMID:22811580

Gene Expression Networks Underlying Ovarian Development in Wild Largemouth Bass (Micropterus salmoides)

PubMed Central

Martyniuk, Christopher J.; Prucha, Melinda S.; Doperalski, Nicholas J.; Antczak, Philipp; Kroll, Kevin J.; Falciani, Francesco; Barber, David S.; Denslow, Nancy D.

2013-01-01

Background Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Methods Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Results Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. Conclusions This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation. PMID:23527095

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

PubMed Central

Zhang, Shan-Shan; Shaw, Robin M.

2016-01-01

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell – cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes. PMID:24460200

Evolution and Expression of Tissue Globins in Ray-Finned Fishes

PubMed Central

Gallagher, Michael D.

2017-01-01

The globin gene family encodes oxygen-binding hemeproteins conserved across the major branches of multicellular life. The origins and evolutionary histories of complete globin repertoires have been established for many vertebrates, but there remain major knowledge gaps for ray-finned fish. Therefore, we used phylogenetic, comparative genomic and gene expression analyses to discover and characterize canonical “non-blood” globin family members (i.e., myoglobin, cytoglobin, neuroglobin, globin-X, and globin-Y) across multiple ray-finned fish lineages, revealing novel gene duplicates (paralogs) conserved from whole genome duplication (WGD) and small-scale duplication events. Our key findings were that: (1) globin-X paralogs in teleosts have been retained from the teleost-specific WGD, (2) functional paralogs of cytoglobin, neuroglobin, and globin-X, but not myoglobin, have been conserved from the salmonid-specific WGD, (3) triplicate lineage-specific myoglobin paralogs are conserved in arowanas (Osteoglossiformes), which arose by tandem duplication and diverged under positive selection, (4) globin-Y is retained in multiple early branching fish lineages that diverged before teleosts, and (5) marked variation in tissue-specific expression of globin gene repertoires exists across ray-finned fish evolution, including several previously uncharacterized sites of expression. In this respect, our data provide an interesting link between myoglobin expression and the evolution of air breathing in teleosts. Together, our findings demonstrate great-unrecognized diversity in the repertoire and expression of nonblood globins that has arisen during ray-finned fish evolution. PMID:28173090

Tc-knirps plays different roles in the specification of antennal and mandibular parasegment boundaries and is regulated by a pair-rule gene in the beetle Tribolium castaneum

PubMed Central

2013-01-01

Background The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function. Results By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is distinct in these segments: Tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary. Surprisingly, Tc-kni controls the timing of expression of the Hox gene Tc-labial in the intercalary segment, although this segment does form in the absence of Tc-kni function. Unexpectedly, we find that the pair-rule gene Tc-even-skipped helps set the posterior boundary of Tc-kni expression in the mandible. Using the mutant antennaless, a likely regulatory Null mutation at the Tc-kni locus, we provide evidence that our RNAi studies represent a Null situation. Conclusions Tc-kni is required for the initiation of the antennal and the maintenance of the mandibular parasegmental boundaries. Tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that Tc-kni is not a canonical ‘gap-gene’. Our finding that a gap gene orthologue is regulated by a pair rule gene adds to the view that the segmentation gene hierarchies differ between Tribolium and Drosophila upstream of the pair rule gene level. In Tribolium, as in Drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving Tc-kni as novel component. PMID:23777260

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits.

PubMed

Zermiani, Monica; Zonin, Elisabetta; Nonis, Alberto; Begheldo, Maura; Ceccato, Luca; Vezzaro, Alice; Baldan, Barbara; Trentin, Annarita; Masi, Antonio; Pegoraro, Marco; Fadanelli, Livio; Teale, William; Palme, Klaus; Quintieri, Luigi; Ruperti, Benedetto

2015-12-01

Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. 'Superficial scald' of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002

DOE Office of Scientific and Technical Information (OSTI.GOV)

Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E.

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system hadmore » a 48-fold dynamic range and was shown to out-perform P trc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.« less

Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002

DOE PAGES

Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E.; ...

2014-09-12

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system hadmore » a 48-fold dynamic range and was shown to out-perform P trc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.« less

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

A dual role for nanos and pumilio in anterior and posterior blastodermal patterning of the short-germ beetle Tribolium castaneum.

PubMed

Schmitt-Engel, Christian; Cerny, Alexander C; Schoppmeier, Michael

2012-04-15

Abdominal patterning in Drosophila requires the function of Nanos (nos) and Pumilio (pum) to repress posterior translation of hunchback mRNA. Here we provide the first functional analysis of nanos and pumilio genes during blastodermal patterning of a short-germ insect. We found that nos and pum in the red flour beetle Tribolium castaneum crucially contribute to posterior segmentation by preventing hunchback translation. While this function seems to be conserved among insects, we provide evidence that Nos and Pum may also act on giant expression, another gap gene. After depletion of nos and pum by parental RNAi, Hunchback and giant remain ectopically at the posterior blastoderm and the posterior Krüppel (Kr) domain is not being activated. giant may be a direct target of Nanos and Pumilio in Tribolium and presumably prevents early Kr expression. In the absence of Kr, the majority of secondary gap gene domains fail to be activated, and abdominal segmentation is terminated prematurely. Surprisingly, we found Nos and Pum also to be involved in early head patterning, as the loss of Nos and Pum results in deletions and transformations of gnathal and pre-gnathal anlagen. Since the targets of Nos and Pum in head development remain to be identified, we propose that anterior patterning in Tribolium may involve additional maternal factors. Copyright © 2012 Elsevier Inc. All rights reserved.

Biolistic transformation of Scoparia dulcis L.

PubMed

Srinivas, Kota; Muralikrishna, Narra; Kumar, Kalva Bharath; Raghu, Ellendula; Mahender, Aileni; Kiranmayee, Kasula; Yashodahara, Velivela; Sadanandam, Abbagani

2016-01-01

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

PubMed Central

Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

2017-01-01

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Lignin, mitochondrial family, and photorespiratory transporter classification as case studies in using co-expression, co-response, and protein locations to aid in identifying transport functions

PubMed Central

Tohge, Takayuki; Fernie, Alisdair R.

2014-01-01

Whole genome sequencing and the relative ease of transcript profiling have facilitated the collection and data warehousing of immense quantities of expression data. However, a substantial proportion of genes are not yet functionally annotated a problem which is particularly acute for transport proteins. In Arabidopsis, for example, only a minor fraction of the estimated 700 intracellular transporters have been identified at the molecular genetic level. Furthermore it is only within the last couple of years that critical genes such as those encoding the final transport step required for the long distance transport of sucrose and the first transporter of the core photorespiratory pathway have been identified. Here we will describe how transcriptional coordination between genes of known function and non-annotated genes allows the identification of putative transporters on the premise that such co-expressed genes tend to be functionally related. We will additionally extend this to include the expansion of this approach to include phenotypic information from other levels of cellular organization such as proteomic and metabolomic data and provide case studies wherein this approach has successfully been used to fill knowledge gaps in important metabolic pathways and physiological processes. PMID:24672529

Novel insights into freshwater hydrocarbon-rich sediments using metatranscriptomics: Opening the black box.

PubMed

Reid, Thomas; Chaganti, Subba Rao; Droppo, Ian G; Weisener, Christopher G

2018-06-01

Baseline biogeochemical surveys of natural environments is an often overlooked field of environmental studies. Too often research begins once contamination has occurred, with a knowledge gap as to how the affected area behaved prior to outside (often anthropogenic) influences. These baseline characterizations can provide insight into proposed bioremediation strategies crucial in cleaning up chemical spill sites or heavily mined regions. Hence, this study was conducted to survey the in-situ microbial activity within freshwater hydrocarbon-rich environments cutting through the McMurray formation - the geologic strata constituting the oil sands. We are the first to report in-situ functional variations among these freshwater microbial ecosystems using metatranscriptomics, providing insight into the in-situ gene expression within these naturally hydrocarbon-rich sites. Key genes involved in energy metabolism (nitrogen, sulfur and methane) and hydrocarbon degradation, including transcripts relating to the observed expression of methane oxidation are reported. This information provides better linkages between hydrocarbon impacted environments, closing knowledge gaps for optimizing not only oil sands mine reclamation but also enhancing microbial reclamation strategies in various freshwater environments. These finding can also be applied to existing contaminated environments, in need of efficient reclamation efforts. Copyright © 2018 Elsevier Ltd. All rights reserved.

An informatics approach to integrating genetic and neurological data in speech and language neuroscience.

PubMed

Bohland, Jason W; Myers, Emma M; Kim, Esther

2014-01-01

A number of heritable disorders impair the normal development of speech and language processes and occur in large numbers within the general population. While candidate genes and loci have been identified, the gap between genotype and phenotype is vast, limiting current understanding of the biology of normal and disordered processes. This gap exists not only in our scientific knowledge, but also in our research communities, where genetics researchers and speech, language, and cognitive scientists tend to operate independently. Here we describe a web-based, domain-specific, curated database that represents information about genotype-phenotype relations specific to speech and language disorders, as well as neuroimaging results demonstrating focal brain differences in relevant patients versus controls. Bringing these two distinct data types into a common database ( http://neurospeech.org/sldb ) is a first step toward bringing molecular level information into cognitive and computational theories of speech and language function. One bridge between these data types is provided by densely sampled profiles of gene expression in the brain, such as those provided by the Allen Brain Atlases. Here we present results from exploratory analyses of human brain gene expression profiles for genes implicated in speech and language disorders, which are annotated in our database. We then discuss how such datasets can be useful in the development of computational models that bridge levels of analysis, necessary to provide a mechanistic understanding of heritable language disorders. We further describe our general approach to information integration, discuss important caveats and considerations, and offer a specific but speculative example based on genes implicated in stuttering and basal ganglia function in speech motor control.

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

PubMed

Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

2013-07-01

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish*

PubMed Central

Hirata, Hiromi; Wen, Hua; Kawakami, Yu; Naganawa, Yuriko; Ogino, Kazutoyo; Yamada, Kenta; Saint-Amant, Louis; Low, Sean E.; Cui, Wilson W.; Zhou, Weibin; Sprague, Shawn M.; Asakawa, Kazuhide; Muto, Akira; Kawakami, Koichi; Kuwada, John Y.

2012-01-01

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca2+ transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers. PMID:22075003

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA

PubMed Central

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5′ and 3′ untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

The receptive-expressive gap in the vocabulary of young second-language learners: Robustness and possible mechanisms.

PubMed

Gibson, Todd A; Oller, D Kimbrough; Jarmulowicz, Linda; Ethington, Corinna A

2012-01-01

Adults and children learning a second language show difficulty accessing expressive vocabulary that appears accessible receptively in their first language (L1). We call this discrepancy the receptive-expressive gap. Kindergarten Spanish (L1) - English (L2) sequential bilinguals were given standardized tests of receptive and expressive vocabulary in both Spanish and English. We found a small receptive-expressive gap in English but a large receptive-expressive gap in Spanish. We categorized children as having had high or low levels of English exposure based on demographic variables and found that the receptive-expressive gap persisted across both levels of English exposure. Regression analyses revealed that variables predicting both receptive and expressive vocabulary scores failed to predict the receptive-expressive gap. The results suggest that the onset of the receptive-expressive gap in L1 must have been abrupt. We discuss possible mechanisms underlying the phenomenon.

The receptive-expressive gap in the vocabulary of young second-language learners: Robustness and possible mechanisms

PubMed Central

Gibson, Todd A.; Oller, D. Kimbrough; Jarmulowicz, Linda; Ethington, Corinna A.

2010-01-01

Adults and children learning a second language show difficulty accessing expressive vocabulary that appears accessible receptively in their first language (L1). We call this discrepancy the receptive-expressive gap. Kindergarten Spanish (L1) - English (L2) sequential bilinguals were given standardized tests of receptive and expressive vocabulary in both Spanish and English. We found a small receptive-expressive gap in English but a large receptive-expressive gap in Spanish. We categorized children as having had high or low levels of English exposure based on demographic variables and found that the receptive-expressive gap persisted across both levels of English exposure. Regression analyses revealed that variables predicting both receptive and expressive vocabulary scores failed to predict the receptive-expressive gap. The results suggest that the onset of the receptive-expressive gap in L1 must have been abrupt. We discuss possible mechanisms underlying the phenomenon. PMID:22247648

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

PubMed Central

Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

2009-01-01

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases.

PubMed

Jain, Ruchi; Dey, Bappaditya; Tyagi, Anil K

2012-10-02

The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model. By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection. We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.

Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model

PubMed Central

Simão, Daniel; Pinto, Catarina; Fernandes, Paulo; Peddie, Christopher J.; Piersanti, Stefania; Collinson, Lucy M.; Salinas, Sara; Saggio, Isabella; Schiavo, Giampietro; Kremer, Eric J.; Brito, Catarina; Alves, Paula M.

2017-01-01

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in pre-clinical tests. For clinical translation, in-depth pre-clinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, while human adenovirus type 5 (HAdV5) showed increased tropism towards glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity. PMID:26181626

Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

PubMed

Simão, D; Pinto, C; Fernandes, P; Peddie, C J; Piersanti, S; Collinson, L M; Salinas, S; Saggio, I; Schiavo, G; Kremer, E J; Brito, C; Alves, P M

2016-01-01

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Identification and characterization of nuclear genes involved in photosynthesis in Populus

PubMed Central

2014-01-01

Background The gap between the real and potential photosynthetic rate under field conditions suggests that photosynthesis could potentially be improved. Nuclear genes provide possible targets for improving photosynthetic efficiency. Hence, genome-wide identification and characterization of the nuclear genes affecting photosynthetic traits in woody plants would provide key insights on genetic regulation of photosynthesis and identify candidate processes for improvement of photosynthesis. Results Using microarray and bulked segregant analysis strategies, we identified differentially expressed nuclear genes for photosynthesis traits in a segregating population of poplar. We identified 515 differentially expressed genes in this population (FC ≥ 2 or FC ≤ 0.5, P < 0.05), 163 up-regulated and 352 down-regulated. Real-time PCR expression analysis confirmed the microarray data. Singular Enrichment Analysis identified 48 significantly enriched GO terms for molecular functions (28), biological processes (18) and cell components (2). Furthermore, we selected six candidate genes for functional examination by a single-marker association approach, which demonstrated that 20 SNPs in five candidate genes significantly associated with photosynthetic traits, and the phenotypic variance explained by each SNP ranged from 2.3% to 12.6%. This revealed that regulation of photosynthesis by the nuclear genome mainly involves transport, metabolism and response to stimulus functions. Conclusions This study provides new genome-scale strategies for the discovery of potential candidate genes affecting photosynthesis in Populus, and for identification of the functions of genes involved in regulation of photosynthesis. This work also suggests that improving photosynthetic efficiency under field conditions will require the consideration of multiple factors, such as stress responses. PMID:24673936

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.

PubMed

Smalheiser, Neil R; Lugli, Giovanni; Thimmapuram, Jyothi; Cook, Edwin H; Larson, John

2011-01-01

We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

PubMed Central

1993-01-01

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer. PMID:8391000

An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

PubMed

Branny, P; de la Torre, F; Garel, J R

1998-04-01

The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.

The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation.

PubMed

Ciovacco, Wendy A; Goldberg, Carolyn G; Taylor, Amanda F; Lemieux, Justin M; Horowitz, Mark C; Donahue, Henry J; Kacena, Melissa A

2009-01-01

Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether this response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18alpha-glycyrrhetinic acid (alphaGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.

The Role of Gap Junctions in Megakaryocyte-Mediated Osteoblast Proliferation and Differentiation

PubMed Central

Ciovacco, Wendy A.; Goldberg, Carolyn G.; Taylor, Amanda F.; Lemieux, Justin M.; Horowitz, Mark C.; Donahue, Henry J.; Kacena, Melissa A.

2009-01-01

Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether that response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18α-glycyrrhetinic acid (αGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MK to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation. PMID:18848655

Immunologic and gene expression profiles of spontaneous canine oligodendrogliomas.

PubMed

Filley, Anna; Henriquez, Mario; Bhowmik, Tanmoy; Tewari, Brij Nath; Rao, Xi; Wan, Jun; Miller, Margaret A; Liu, Yunlong; Bentley, R Timothy; Dey, Mahua

2018-05-01

Malignant glioma (MG), the most common primary brain tumor in adults, is extremely aggressive and uniformly fatal. Several treatment strategies have shown significant preclinical promise in murine models of glioma; however, none have produced meaningful clinical responses in human patients. We hypothesize that introduction of an additional preclinical animal model better approximating the complexity of human MG, particularly in interactions with host immune responses, will bridge the existing gap between these two stages of testing. Here, we characterize the immunologic landscape and gene expression profiles of spontaneous canine glioma and evaluate its potential for serving as such a translational model. RNA in situ hybridization, flowcytometry, and RNA sequencing were used to evaluate immune cell presence and gene expression in healthy and glioma-bearing canines. Similar to human MGs, canine gliomas demonstrated increased intratumoral immune cell infiltration (CD4+, CD8+ and CD4+Foxp3+ T cells). The peripheral blood of glioma-bearing dogs also contained a relatively greater proportion of CD4+Foxp3+ regulatory T cells and plasmacytoid dendritic cells. Tumors were strongly positive for PD-L1 expression and glioma-bearing animals also possessed a greater proportion of immune cells expressing the immune checkpoint receptors CTLA-4 and PD-1. Analysis of differentially expressed genes in our canine populations revealed several genetic changes paralleling those known to occur in human disease. Naturally occurring canine glioma has many characteristics closely resembling human disease, particularly with respect to genetic dysregulation and host immune responses to tumors, supporting its use as a translational model in the preclinical testing of prospective anti-glioma therapies proven successful in murine studies.

Hydrodynamics based transfection in normal and fibrotic rats

PubMed Central

Yeikilis, Rita; Gal, Shunit; Kopeiko, Natalia; Paizi, Melia; Pines, Mark; Braet, Filip; Spira, Gadi

2006-01-01

AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect. CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced. PMID:17036386

Development of a versatile enrichment analysis tool reveals associations between the maternal brain and mental health disorders, including autism

PubMed Central

2013-01-01

Background A recent study of lateral septum (LS) suggested a large number of autism-related genes with altered expression in the postpartum state. However, formally testing the findings for enrichment of autism-associated genes proved to be problematic with existing software. Many gene-disease association databases have been curated which are not currently incorporated in popular, full-featured enrichment tools, and the use of custom gene lists in these programs can be difficult to perform and interpret. As a simple alternative, we have developed the Modular Single-set Enrichment Test (MSET), a minimal tool that enables one to easily evaluate expression data for enrichment of any conceivable gene list of interest. Results The MSET approach was validated by testing several publicly available expression data sets for expected enrichment in areas of autism, attention deficit hyperactivity disorder (ADHD), and arthritis. Using nine independent, unique autism gene lists extracted from association databases and two recent publications, a striking consensus of enrichment was detected within gene expression changes in LS of postpartum mice. A network of 160 autism-related genes was identified, representing developmental processes such as synaptic plasticity, neuronal morphogenesis, and differentiation. Additionally, maternal LS displayed enrichment for genes associated with bipolar disorder, schizophrenia, ADHD, and depression. Conclusions The transition to motherhood includes the most fundamental social bonding event in mammals and features naturally occurring changes in sociability. Some individuals with autism, schizophrenia, or other mental health disorders exhibit impaired social traits. Genes involved in these deficits may also contribute to elevated sociability in the maternal brain. To date, this is the first study to show a significant, quantitative link between the maternal brain and mental health disorders using large scale gene expression data. Thus, the postpartum brain may provide a novel and promising platform for understanding the complex genetics of improved sociability that may have direct relevance for multiple psychiatric illnesses. This study also provides an important new tool that fills a critical analysis gap and makes evaluation of enrichment using any database of interest possible with an emphasis on ease of use and methodological transparency. PMID:24245670

Development of a versatile enrichment analysis tool reveals associations between the maternal brain and mental health disorders, including autism.

PubMed

Eisinger, Brian E; Saul, Michael C; Driessen, Terri M; Gammie, Stephen C

2013-11-19

A recent study of lateral septum (LS) suggested a large number of autism-related genes with altered expression in the postpartum state. However, formally testing the findings for enrichment of autism-associated genes proved to be problematic with existing software. Many gene-disease association databases have been curated which are not currently incorporated in popular, full-featured enrichment tools, and the use of custom gene lists in these programs can be difficult to perform and interpret. As a simple alternative, we have developed the Modular Single-set Enrichment Test (MSET), a minimal tool that enables one to easily evaluate expression data for enrichment of any conceivable gene list of interest. The MSET approach was validated by testing several publicly available expression data sets for expected enrichment in areas of autism, attention deficit hyperactivity disorder (ADHD), and arthritis. Using nine independent, unique autism gene lists extracted from association databases and two recent publications, a striking consensus of enrichment was detected within gene expression changes in LS of postpartum mice. A network of 160 autism-related genes was identified, representing developmental processes such as synaptic plasticity, neuronal morphogenesis, and differentiation. Additionally, maternal LS displayed enrichment for genes associated with bipolar disorder, schizophrenia, ADHD, and depression. The transition to motherhood includes the most fundamental social bonding event in mammals and features naturally occurring changes in sociability. Some individuals with autism, schizophrenia, or other mental health disorders exhibit impaired social traits. Genes involved in these deficits may also contribute to elevated sociability in the maternal brain. To date, this is the first study to show a significant, quantitative link between the maternal brain and mental health disorders using large scale gene expression data. Thus, the postpartum brain may provide a novel and promising platform for understanding the complex genetics of improved sociability that may have direct relevance for multiple psychiatric illnesses. This study also provides an important new tool that fills a critical analysis gap and makes evaluation of enrichment using any database of interest possible with an emphasis on ease of use and methodological transparency.

Interactive effects of inflammatory cytokine and abundant low-molecular-weight PAHs on inhibition of gap junctional intercellular communication, disruption of cell proliferation control, and the AhR-dependent transcription.

PubMed

Kabátková, Markéta; Svobodová, Jana; Pěnčíková, Kateřina; Mohatad, Dilshad Shaik; Šmerdová, Lenka; Kozubík, Alois; Machala, Miroslav; Vondráček, Jan

2015-01-05

Polycyclic aromatic hydrocarbons (PAHs) with lower molecular weight exhibit lesser genotoxicity and carcinogenicity than highly carcinogenic PAHs with a higher number of benzene rings. Nevertheless, they elicit specific effects linked with tumor promotion, such as acute inhibition of gap junctional intercellular communication (GJIC). Although inflammatory reaction may alter bioactivation and toxicity of carcinogenic PAHs, little is known about the impact of pro-inflammatory cytokines on toxic effects of the low-molecular-weight PAHs. Here, we investigated the impact of a pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), on the effects associated with tumor promotion and with induction of the aryl hydrocarbon receptor (AhR)-dependent gene expression in rat liver epithelial cells. We found that a prolonged incubation with TNF-α induced a down-regulation of GJIC, associated with reduced expression of connexin 43 (Cx43), a major connexin isoform found in liver epithelial cells. The Cx43 down-regulation was partly mediated by the activity of the mitogen-activated protein (MAP) p38 kinase. Independently of GJIC modulation, or p38 activation, TNF-α potentiated the AhR-dependent proliferative effect of a model low-molecular-weight PAH, fluoranthene, on contact-inhibited cells. In contrast, this pro-inflammatory cytokine repressed the fluoranthene-induced expression of a majority of model AhR gene targets, such as Cyp1a1, Ahrr or Tiparp. The results of the present study indicate that inflammatory reaction may differentially modulate various toxic effects of low-molecular-weight PAHs; the exposure to pro-inflammatory cytokines may both strengthen (inhibition of GJIC, disruption of contact inhibition) and repress (expression of a majority of AhR-dependent genes) their impact on toxic endpoints associated with carcinogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Ruttenstock, Elke Maria; Doi, Takashi; Dingemann, Jens; Puri, Prem

2012-02-01

Connexin 43 (Cx43), a major gap junction protein, is necessary for alveologenesis and plays an important role in the differentiation of type II to type I alveolar epithelial cells. Knockout mice of Cx43 display severe pulmonary hypoplasia (PH). Prenatal administration of retinoic acid (RA) is known to stimulate alveologenesis in nitrofen-induced PH. Recent studies revealed that retinoids upregulate Cx43 expression. We hypothesized that gene expression of Cx43 is downregulated during alveologenesis and that administration of RA upregulates Cx43 expression in the nitrofen-induced PH. Pregnant rats were exposed to olive oil or nitrofen on day 9 (D9) of gestation. Retinoic acid was given intraperitoneally on D18, D19, and D20. Fetal lungs were harvested on D18 and D21 and divided into control, nitrofen, control+RA (D21), and nitrofen+RA (D21). The Cx43 expression levels were determined using reverse transcription polymerase chain reaction and immunohistochemistry. On D18 and D21, Cx43 relative messenger RNA expression levels were significantly downregulated in nitrofen compared with those in the control group. On D21, expression levels of Cx43 were significantly upregulated in nitrofen+RA and control+RA compared with those in nitrofen group. Immunohistochemical studies confirmed these results. Downregulation of Cx43 expression may interfere with normal alveologenesis. Upregulation of Cx43 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in nitrofen-induced PH. Copyright © 2012 Elsevier Inc. All rights reserved.

Tools to minimize interlaboratory variability in vitellogenin gene expression monitoring programs

USGS Publications Warehouse

Jastrow, Aaron; Gordon, Denise A.; Auger, Kasie M.; Punska, Elizabeth C.; Arcaro, Kathleen F.; Keteles, Kristen; Winkelman, Dana L.; Lattier, David; Biales, Adam; Lazorchak, James M.

2017-01-01

The egg yolk precursor protein vitellogenin is widely used as a biomarker of estrogen exposure in male fish. However, standardized methodology is lacking and little is known regarding the reproducibility of results among laboratories using different equipment, reagents, protocols, and data analysis programs. To address this data gap we tested the reproducibility across laboratories to evaluate vitellogenin gene (vtg) expression and assessed the value of using a freely available software data analysis program. Samples collected from studies of male fathead minnows (Pimephales promelas) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent were evaluated for vtg expression in 4 laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis LinRegPCR is used, with 3 of 4 laboratories detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All 4 laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent compared with 15 male fish held in a control stream. Finally, we were able to determine that the source of high interlaboratory variability from complementary deoxyribonucleic acid (cDNA) to quantitative polymerase chain reaction (qPCR) analyses was the expression analysis software unique to each real-time qPCR machine. We successfully eliminated the interlaboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and polymerase chain reaction (PCR) efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs should validate their PCR protocols and analyze their gene expression data following the guidelines established in the present study for all gene expression biomarkers. 

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions.

PubMed

Duary, Raj Kumar; Batish, Virender Kumar; Grover, Sunita

2012-03-01

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

Feeding State, Insulin and NPR-1 Modulate Chemoreceptor Gene Expression via Integration of Sensory and Circuit Inputs

PubMed Central

Gruner, Matthew; Nelson, Dru; Winbush, Ari; Hintz, Rebecca; Ryu, Leesun; Chung, Samuel H.; Kim, Kyuhyung; Gabel, Chrisopher V.; van der Linden, Alexander M.

2014-01-01

Feeding state and food availability can dramatically alter an animals' sensory response to chemicals in its environment. Dynamic changes in the expression of chemoreceptor genes may underlie some of these food and state-dependent changes in chemosensory behavior, but the mechanisms underlying these expression changes are unknown. Here, we identified a KIN-29 (SIK)-dependent chemoreceptor, srh-234, in C. elegans whose expression in the ADL sensory neuron type is regulated by integration of sensory and internal feeding state signals. We show that in addition to KIN-29, signaling is mediated by the DAF-2 insulin-like receptor, OCR-2 TRPV channel, and NPR-1 neuropeptide receptor. Cell-specific rescue experiments suggest that DAF-2 and OCR-2 act in ADL, while NPR-1 acts in the RMG interneurons. NPR-1-mediated regulation of srh-234 is dependent on gap-junctions, implying that circuit inputs regulate the expression of chemoreceptor genes in sensory neurons. Using physical and genetic manipulation of ADL neurons, we show that sensory inputs from food presence and ADL neural output regulate srh-234 expression. While KIN-29 and DAF-2 act primarily via the MEF-2 (MEF2) and DAF-16 (FOXO) transcription factors to regulate srh-234 expression in ADL neurons, OCR-2 and NPR-1 likely act via a calcium-dependent but MEF-2- and DAF-16-independent pathway. Together, our results suggest that sensory- and circuit-mediated regulation of chemoreceptor genes via multiple pathways may allow animals to precisely regulate and fine-tune their chemosensory responses as a function of internal and external conditions. PMID:25357003

Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

PubMed Central

Kinjo, Takahiko; Tanaka, Makoto; Osanai, Tomohiro; Shibutani, Shuji; Narita, Ikuyo; Tanno, Tomohiro; Nishizaki, Kimitaka; Ichikawa, Hiroaki; Kimura, Yoshihiro; Ishida, Yuji; Yokota, Takashi; Shimada, Michiko; Homma, Yoshimi; Tomita, Hirofumi; Okumura, Ken

2015-01-01

Background We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm. Methods and Results We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice. Conclusions VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA. PMID:26624289

The genetics of geometry

PubMed Central

Coen, Enrico; Rolland-Lagan, Anne-Gaëlle; Matthews, Mark; Bangham, J. Andrew; Prusinkiewicz, Przemyslaw

2004-01-01

Although much progress has been made in understanding how gene expression patterns are established during development, much less is known about how these patterns are related to the growth of biological shapes. Here we describe conceptual and experimental approaches to bridging this gap, with particular reference to plant development where lack of cell movement simplifies matters. Growth and shape change in plants can be fully described with four types of regional parameter: growth rate, anisotropy, direction, and rotation. A key requirement is to understand how these parameters both influence and respond to the action of genes. This can be addressed by using mechanistic models that capture interactions among three components: regional identities, regionalizing morphogens, and polarizing morphogens. By incorporating these interactions within a growing framework, it is possible to generate shape changes and associated gene expression patterns according to particular hypotheses. The results can be compared with experimental observations of growth of normal and mutant forms, allowing further hypotheses and experiments to be formulated. We illustrate these principles with a study of snapdragon petal growth. PMID:14960734

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

PubMed

Boonvitthya, Nassapat; Tanapong, Phatrapan; Kanngan, Patcharaporn; Burapatana, Vorakan; Chulalaksananukul, Warawut

2012-10-01

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells

PubMed Central

Wu, Li; Wang, Yuan; Wu, Junzhou; Lv, Cong; Wang, Jie; Tang, Xinjing

2013-01-01

We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies. PMID:23104375

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

PubMed Central

Cohen-Tannoudji, Michel; Robine, Sylvie; Choulika, André; Pinto, Daniel; El Marjou, Fatima; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric

1998-01-01

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest. PMID:9488460

Genome-scale analysis identifies GJB2 and ERO1LB as prognosis markers in patients with pancreatic cancer.

PubMed

Zhu, Tao; Gao, Yuan-Feng; Chen, Yi-Xin; Wang, Zhi-Bin; Yin, Ji-Ye; Mao, Xiao-Yuan; Li, Xi; Zhang, Wei; Zhou, Hong-Hao; Liu, Zhao-Qian

2017-03-28

Pancreatic cancer is a complex and heterogeneous disease with the etiology largely unknown. The deadly nature of pancreatic cancer, with an extremely low 5-year survival rate, renders urgent a better understanding of the molecular events underlying it. The aim of this study is to investigate the gene expression module of pancreatic adenocarcinoma and to identify differentially expressed genes (DEGs) with prognostic potentials. Transcriptome microarray data of five GEO datasets (GSE15471, GSE16515, GSE18670, GSE32676, GSE71989), including 117 primary tumor samples and 73 normal pancreatic tissue samples, were utilized to identify DEGs. The five sets of DEGs had an overlapping subset consisting of 98 genes (90 up-regulated and 8 down-regulated), which were probably common to pancreatic cancer. Gene ontology (GO) analysis of the 98 DEGs showed that cell cycle and cell adhesion were the major enriched processes, and extracellular matrix (ECM)-receptor interaction and p53 signaling pathway were the most enriched pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Elevated expression of gap junction protein beta 2 (GJB2) and reduced endoplasmic reticulum oxidoreductase 1-like beta (ERO1LB) expression were validated in an independent cohort. Kaplan-Meier survival analysis revealed that GJB2 and ERO1LB levels were significantly associated with the overall survival of pancreatic cancer patients. GJB2 and ERO1LB are implicated in pancreatic cancer progression and can be used to predict patient survival. Therapeutic strategies targeting GJB2 and facilitating ERO1LB expression may deserve evaluation to improve prognosis of pancreatic cancer patients.

Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

PubMed

Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

2015-02-01

Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects.

PubMed

Kublin, James G; Mikolajczak, Sebastian A; Sack, Brandon K; Fishbaugher, Matt E; Seilie, Annette; Shelton, Lisa; VonGoedert, Tracie; Firat, Melike; Magee, Sara; Fritzen, Emma; Betz, Will; Kain, Heather S; Dankwa, Dorender A; Steel, Ryan W J; Vaughan, Ashley M; Noah Sather, D; Murphy, Sean C; Kappe, Stefan H I

2017-01-04

Immunization of humans with whole sporozoites confers complete, sterilizing immunity against malaria infection. However, achieving consistent safety while maintaining immunogenicity of whole parasite vaccines remains a formidable challenge. We generated a genetically attenuated Plasmodium falciparum (Pf) malaria parasite by deleting three genes expressed in the pre-erythrocytic stage (Pf p52 - /p36 - /sap1 - ). We then tested the safety and immunogenicity of the genetically engineered (Pf GAP3KO) sporozoites in human volunteers. Pf GAP3KO sporozoites were delivered to 10 volunteers using infected mosquito bites with a single exposure consisting of 150 to 200 bites per subject. All subjects remained blood stage-negative and developed inhibitory antibodies to sporozoites. GAP3KO rodent malaria parasites engendered complete, protracted immunity against infectious sporozoite challenge in mice. The results warrant further clinical testing of Pf GAP3KO and its potential development into a vaccine strain. Copyright © 2017, American Association for the Advancement of Science.

Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival.

PubMed

Laws, Mary J; Taylor, Robert N; Sidell, Neil; DeMayo, Francesco J; Lydon, John P; Gutstein, David E; Bagchi, Milan K; Bagchi, Indrani C

2008-08-01

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

PubMed

Eckardt, D; Theis, M; Degen, J; Ott, T; van Rijen, H V M; Kirchhoff, S; Kim, J-S; de Bakker, J M T; Willecke, K

2004-01-01

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

Deciphering defective amelogenesis using in vitro culture systems.

PubMed

Arinawati, Dian Yosi; Miyoshi, Keiko; Tanimura, Ayako; Horiguchi, Taigo; Hagita, Hiroko; Noma, Takafumi

2018-04-01

The conventional two-dimensional (2D) in vitro culture system is frequently used to analyze the gene expression with or without extracellular signals. However, the cells derived from primary culture and cell lines frequently deviate the gene expression profile compared to the corresponding in vivo samples, which sometimes misleads the actual gene regulation in vivo. To overcome this gap, we developed the comparative 2D and 3D in vitro culture systems and applied them to the genetic study of amelogenesis imperfecta (AI) as a model. Recently, we found specificity protein 6 (Sp6) mutation in an autosomal-recessive AI rat that was previously named AMI. We constructed 3D structure of ARE-B30 cells (AMI-derived rat dental epithelial cells) or G5 (control wild type cells) combined with RPC-C2A cells (rat pulp cell line) separated by the collagen membrane, while in 2D structure, ARE-B30 or G5 was cultured with or without the collagen membrane. Comparative analysis of amelogenesis-related gene expression in ARE-B30 and G5 using our 2D and 3D in vitro systems revealed distinct expression profiles, showing the causative outcomes. Bone morphogenetic protein 2 and follistatin were reciprocally expressed in G5, but not in ARE-B30 cells. All-or-none expression of amelotin, kallikrein-related peptidase 4, and nerve growth factor receptor was observed in both cell types. In conclusion, our in vitro culture systems detected the phenotypical differences in the expression of the stage-specific amelogenesis-related genes. Parallel analysis with 2D and 3D culture systems may provide a platform to understand the molecular basis for defective amelogenesis caused by Sp6 mutation. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

notch3 is essential for oligodendrocyte development and vascular integrity in zebrafish

PubMed Central

Zaucker, Andreas; Mercurio, Sara; Sternheim, Nitzan; Talbot, William S.; Marlow, Florence L.

2013-01-01

SUMMARY Mutations in the human NOTCH3 gene cause CADASIL syndrome (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). CADASIL is an inherited small vessel disease characterized by diverse clinical manifestations including vasculopathy, neurodegeneration and dementia. Here we report two mutations in the zebrafish notch3 gene, one identified in a previous screen for mutations with reduced expression of myelin basic protein (mbp) and another caused by a retroviral insertion. Reduced mbp expression in notch3 mutant embryos is associated with fewer oligodendrocyte precursor cells (OPCs). Despite an early neurogenic phenotype, mbp expression recovered at later developmental stages and some notch3 homozygous mutants survived to adulthood. These mutants, as well as adult zebrafish carrying both mutant alleles together, displayed a striking stress-associated accumulation of blood in the head and fins. Histological analysis of mutant vessels revealed vasculopathy, including: an enlargement (dilation) of vessels in the telencephalon and fin, disorganization of the normal stereotyped arrangement of vessels in the fin, and an apparent loss of arterial morphological structure. Expression of hey1, a well-known transcriptional target of Notch signaling, was greatly reduced in notch3 mutant fins, suggesting that Notch3 acts via a canonical Notch signaling pathway to promote normal vessel structure. Ultrastructural analysis confirmed the presence of dilated vessels in notch3 mutant fins and revealed that the vessel walls of presumed arteries showed signs of deterioration. Gaps in the arterial wall and the presence of blood cells outside of vessels in mutants indicated that compromised vessel structure led to hemorrhage. In notch3 heterozygotes, we found elevated expression of both notch3 itself and target genes, indicating that specific alterations in gene expression due to partial loss of Notch3 function might contribute to the abnormalities observed in heterozygous larvae and adults. Our analysis of zebrafish notch3 mutants indicates that Notch3 regulates OPC development and mbp gene expression in larvae, and maintains vascular integrity in adults. PMID:23720232

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

PubMed Central

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-01-01

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

PubMed

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-03-16

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 ( FGF1 and FGF12 ), bone morphogenetic factor-1 ( BMP1 ), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 ( SMARCA4 ), Matrix extracellular phosphoglycoprotein ( MEPE ), Integrin, β4 ( ITGBP4 ), Matrix Metalloproteinase -1, -28 ( MMP1 and MMP28 ), and signal transducer and activator of transcription-4 ( STAT4 ) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1 , MMP28 and kallikrein related peptidase-7 ( KLK7 ), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

A prior-based integrative framework for functional transcriptional regulatory network inference

PubMed Central

Siahpirani, Alireza F.

2017-01-01

Abstract Transcriptional regulatory networks specify regulatory proteins controlling the context-specific expression levels of genes. Inference of genome-wide regulatory networks is central to understanding gene regulation, but remains an open challenge. Expression-based network inference is among the most popular methods to infer regulatory networks, however, networks inferred from such methods have low overlap with experimentally derived (e.g. ChIP-chip and transcription factor (TF) knockouts) networks. Currently we have a limited understanding of this discrepancy. To address this gap, we first develop a regulatory network inference algorithm, based on probabilistic graphical models, to integrate expression with auxiliary datasets supporting a regulatory edge. Second, we comprehensively analyze our and other state-of-the-art methods on different expression perturbation datasets. Networks inferred by integrating sequence-specific motifs with expression have substantially greater agreement with experimentally derived networks, while remaining more predictive of expression than motif-based networks. Our analysis suggests natural genetic variation as the most informative perturbation for network inference, and, identifies core TFs whose targets are predictable from expression. Multiple reasons make the identification of targets of other TFs difficult, including network architecture and insufficient variation of TF mRNA level. Finally, we demonstrate the utility of our inference algorithm to infer stress-specific regulatory networks and for regulator prioritization. PMID:27794550

Expression of the MDR-1 gene-encoded P-glycoprotein in cardiomyocytes of conscious sheep undergoing acute myocardial ischemia followed by reperfusion.

PubMed

Laguens, Rubén P; Lazarowski, Alberto J; Cuniberti, Luis A; Vera Janavel, Gustavo L; Cabeza Meckert, Patricia M; Yannarelli, Gustavo G; del Valle, Héctor F; Lascano, Elena C; Negroni, Jorge A; Crottogini, Alberto J

2007-02-01

We have recently reported that in chronic myocardial ischemia, adult mammalian cardiomyocytes express P-glycoprotein (P-gp). We now investigate if P-gp is also expressed in acute regional ischemia followed by reperfusion. Adult conscious sheep underwent 12-min occlusion of the mid-left anterior descending artery (inflatable cuff). Successful ischemia-reperfusion was confirmed by monitoring percent systolic left ventricular anterior wall thickening (sonomicrometry) during the whole ischemic period and every 10 min over 2 hr following cuff deflation. At 3, 24, and 48 hr after reperfusion, P-gp expression was investigated by immunohistochemistry and Western blot and MDR-1 mRNA by RT-PCR. Cardiomyocytes in the occluded artery territory (but not those in remote areas) consistently expressed P-gp at their sarcolemma. Whereas at 3 and 24 hr P-gp was mainly observed in the T tubules, at 48 hr it predominated in intercalated discs and gap junctions. RT-PCR and Western blot revealed higher expression in ischemic than in control myocardium. We conclude that in adult sheep with acute myocardial ischemia, the MDR-1 gene-encoded P-gp is expressed at the sarcolemma of the cardiomyocytes from 3 hr up to at least 48 hr after reperfusion.

The effect of titanium dioxide nanoparticles on antioxidant gene expression in tilapia ( Oreochromis niloticus)

NASA Astrophysics Data System (ADS)

Varela-Valencia, Ruth; Gómez-Ortiz, Nikte; Oskam, Gerko; de Coss, Romeo; Rubio-Piña, Jorge; del Río-García, Marcela; Albores-Medina, Arnulfo; Zapata-Perez, Omar

2014-04-01

The reactivity of nanoparticles (NPs) in biological systems is well recognized, but there are huge gaps in our understanding of NP toxicity in fish, despite a number of recent ecotoxicity studies. Therefore, the aim of this research was to evaluate the effect of titanium dioxide NPs (TiO2-NPs) on antioxidant gene expression in the tilapia, Oreochromis niloticus. First, different sizes, shapes, and phases of TiO2-NPs were synthesized and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS). Fish were injected intraperitoneally with different concentrations (0.1, 1.0, 10.0 mg/L), sizes (7, 14, and 21 nm), and phases (anatase and rutile) of TiO2-NPs, and sacrificed 3, 6, 12, and 24 h after injection, when their livers were removed. Total RNA was extracted, and expression of the catalase ( CAT), glutathione- S-transferase ( GST), and superoxide dismutase ( SOD) genes was assessed by real-time polymerase chain reaction (RT-PCR). The results showed that injection of 1.0 mg/L TiO2-NPs induced an initial mild increase in CAT, GST, and SOD gene expression in tilapia, after which transcript levels decreased. Fish injected with 7 and 14 nm TiO2-NPs showed an increase in antioxidant transcript levels 6 h after treatment. Finally, the rutile form generated stronger induction of the GST gene than anatase TiO2-NPs during the first 6 h after injection, which suggests that exposure to rutile causes higher levels of reactive oxygen species to be produced.

Insecticide imidacloprid influences cognitive functions and alters learning performance and related gene expression in a rat model.

PubMed

Kara, Murat; Yumrutas, Onder; Demir, Caner F; Ozdemir, Hasan Huseyin; Bozgeyik, Ibrahim; Coskun, Salih; Eraslan, Ersen; Bal, Ramazan

2015-10-01

The potential toxic effects of several pesticides, including imidacloprid on non-target organisms have not been clearly established. Also, the chronic effects of non-toxic doses on cognitive function in mammals are unknown. In this study, the effects of different doses of imidacloprid on learning and memory of infant and adult rats were evaluated, and the expressions of genes synthesizing proteins known to be associated with learning in brain tissues were also documented. 0.5, 2 and 8 mg/kg doses of imidacloprid were administered to newborn infant and adult Wistar albino rats by gavage. Their learning activities were evaluated, and the expression levels of the inotropic glutamate receptor GRIN1, synoptophysin, growth-associated protein 43 and the muscarinic receptor M1 in hippocampus were determined by real-time PCR method. Learning activities were diminished significantly at 2 and 8 mg/kg doses in the infant model groups and at 8 mg/kg dose in adult rats. Also, expression levels of GRIN1, SYP and GAP-43 were found to be insignificantly altered. Only the expression of M1 were significantly changed in high doses of adult group. Thus imidacloprid in high doses causes deterioration in cognitive functions particularly in infant rats, and this deterioration may be associated with changes in the expressions of related genes. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

ARHGAP18 is a novel gene under positive natural selection that influences HbF levels in β-thalassaemia.

PubMed

He, Yunyan; Luo, Jianming; Chen, Yang; Zhou, Xiaoheng; Yu, Shanjuan; Jin, Ling; Xiao, Xuan; Jia, Siyuan; Liu, Qiang

2018-02-01

Foetal haemoglobin (HbF) plays a dominant role in ameliorating the morbidity and mortality of β-thalassaemia. A better understanding of the loci and genes involved in HbF expression would be beneficial for the treatment of β-thalassaemia major. However, the genes associated with HbF expression remain largely unknown. In this study, we first explored large-scale data sets and examined the human genome for evidence of positive natural selection to screen out single nucleotide polymorphisms (SNPs). A genetic analysis of HbF levels was conducted in a Chinese cohort of patients with β-thalassaemia to confirm the bioinformatics results. A total of 1141 subjects with β-thalassaemia were recruited. The results showed that the SNP rs11759328 in the ARHGAP18 gene was significantly associated with HbF levels (Ρ = 5.1 × 10 -4 ). ARHGAP18 belongs to the RhoGAP family and controls angiogenesis, cellular morphology and motility. Second, after determining that ARHGAP18 was highly expressed in the human K562 cell line, we used lentiviral-mediated small interfering RNA to knock down ARHGAP18 expression and subsequently assessed cell proliferation and apoptosis using cell proliferation assays and flow cytometry, respectively. ARHGAP18 downregulation in K562 cells significantly increased HBG1/2 expression and apoptosis, but proliferation was not significantly affected in vitro. Our data suggest that ARHGAP18, which was located by the SNP rs11759328 via positive selection, plays a potential role in regulating HbF expression in β-thalassaemia and may be a promising therapeutic target. Knockout studies of ARHGAP18 warrant further investigation into its aetiology in HbF.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.

PubMed

Jang, Heeun; Levy, Sagi; Flavell, Steven W; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I

2017-02-14

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9 -based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans

PubMed Central

Jang, Heeun; Levy, Sagi; Flavell, Steven W.; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I.

2017-01-01

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans. The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9–containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9–based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits. PMID:28143932

Evolution of Drosophila ribosomal protein gene core promoters.

PubMed

Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

2009-03-01

The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

Evolution of Drosophila ribosomal protein gene core promoters

PubMed Central

Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

2011-01-01

The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

Transcription Analysis of the Myometrium of Labouring and Non-Labouring Women

PubMed Central

Hutchinson, James L.; Hibbert, Nanette; Freeman, Tom C.; Saunders, Philippa T. K.; Norman, Jane E.

2016-01-01

An incomplete understanding of the molecular mechanisms that initiate normal human labour at term seriously hampers the development of effective ways to predict, prevent and treat disorders such as preterm labour. Appropriate analysis of large microarray experiments that compare gene expression in non-labouring and labouring gestational tissues is necessary to help bridge these gaps in our knowledge. In this work, gene expression in 48 (22 labouring, 26 non-labouring) lower-segment myometrial samples collected at Caesarean section were analysed using Illumina HT-12 v4.0 BeadChips. Normalised data were compared between labouring and non-labouring groups using traditional statistical methods and a novel network graph approach. We sought technical validation with quantitative real-time PCR, and biological replication through inverse variance-weighted meta-analysis with published microarray data. We have extended the list of genes suggested to be associated with labour: Compared to non-labouring samples, labouring samples showed apparent higher expression at 960 probes (949 genes) and apparent lower expression at 801 probes (789 genes) (absolute fold change ≥1.2, rank product percentage of false positive value (RP-PFP) <0.05). Although half of the women in the labouring group had received pharmaceutical treatment to induce or augment labour, sensitivity analysis suggested that this did not confound our results. In agreement with previous studies, functional analysis suggested that labour was characterised by an increase in the expression of inflammatory genes and network analysis suggested a strong neutrophil signature. Our analysis also suggested that labour is characterised by a decrease in the expression of muscle-specific processes, which has not been explicitly discussed previously. We validated these findings through the first formal meta-analysis of raw data from previous experiments and we hypothesise that this represents a change in the composition of myometrial tissue at labour. Further work will be necessary to reveal whether these results are solely due to leukocyte infiltration into the myometrium as a mechanism initiating labour, or in addition whether they also represent gene changes in the myocytes themselves. We have made all our data available at www.ebi.ac.uk/arrayexpress/ (accession number E-MTAB-3136) to facilitate progression of this work. PMID:27176052

Analysis of lead toxicity in human cells.

PubMed

Gillis, Bruce S; Arbieva, Zarema; Gavin, Igor M

2012-07-27

Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels. The results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies.

Analysis of lead toxicity in human cells

PubMed Central

2012-01-01

Background Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. Results We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels. Conclusions The results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies. PMID:22839698

The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models

PubMed Central

Schrobback, Karsten; Klein, Travis Jacob

2015-01-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell–cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell–cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5′-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell–cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs. PMID:25693425

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

PubMed

Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

2015-06-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Identification of polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gindhart, J.G. Jr.; Kaufman, T.C.

1995-02-01

The Drosophilia homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products.more » When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of Polycomb and trithorax group loci. 98 refs., 6 figs., 4 tabs.« less

Monocytic cell junction proteins serve important roles in atherosclerosis via the endoglin pathway

PubMed Central

Chen, Lina; Chen, Zhongliang; Ge, Menghua; Tang, Oushan; Cheng, Yinhong; Zhou, Haoliang; Shen, Yu; Qin, Fengming

2017-01-01

The formation of atherosclerosis is recognized to be caused by multiple factors including pathogenesis in monocytes during inflammation. The current study provided evidence that monocytic junctions were significantly altered in patients with atherosclerosis, which suggested an association between cell junctions and atherosclerosis. Claudin-1, occludin-1 and ZO-1 were significantly enhanced in atherosclerosis, indicating that the tight junction pathway was activated during the pathogenesis of atherosclerosis. In addition, the gene expression of 5 connexin members involved in the gap junction pathway were quantified, indicating that connexin 43 and 46 were significantly up-regulated in atherosclerosis. Furthermore, inflammatory factors including endoglin and SMAD were observed, suggesting that immune regulative factors were down-regulated in this pathway. Silicon-based analysis additionally identified that connexins and tight junctions were altered in association with monocytic inflammation regulations, endoglin pathway. The results imply that reduced expression of the immune regulation pathway in monocytes is correlated with the generation of gap junctions and tight junctions which serve important roles in atherosclerosis. PMID:28901429

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

PubMed

Liddelow, Shane A; Dziegielewska, Katarzyna M; Ek, C Joakim; Habgood, Mark D; Bauer, Hannelore; Bauer, Hans-Christian; Lindsay, Helen; Wakefield, Matthew J; Strazielle, Nathalie; Kratzer, Ingrid; Møllgård, Kjeld; Ghersi-Egea, Jean-François; Saunders, Norman R

2013-01-01

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Gap junction coupling is required for tumor cell migration through lymphatic endothelium.

PubMed

Karpinich, Natalie O; Caron, Kathleen M

2015-05-01

The lymphatic vasculature is a well-established conduit for metastasis, but the mechanisms by which tumor cells interact with lymphatic endothelial cells (LECs) to facilitate escape remain poorly understood. Elevated levels of the lymphangiogenic peptide adrenomedullin are found in many tumors, and we previously characterized that its expression is necessary for lymphatic vessel growth within both tumors and sentinel lymph nodes and for distant metastasis. This study used a tumor cell-LEC coculture system to identify a series of adrenomedullin-induced events that facilitated transendothelial migration of the tumor cells through a lymphatic monolayer. High levels of adrenomedullin expression enhanced adhesion of tumor cells to LECs, and further analysis revealed that adrenomedullin promoted gap junction coupling between LECs as evidenced by spread of Lucifer yellow dye. Adrenomedullin also enhanced heterocellular gap junction coupling as demonstrated by Calcein dye transfer from tumor cells into LECs. This connexin-mediated gap junction intercellular communication was necessary for tumor cells to undergo transendothelial migration because pharmacological blockade of this heterocellular communication prevented the ability of tumor cells to transmigrate through the lymphatic monolayer. In addition, treatment of LECs with adrenomedullin caused nuclear translocation of β-catenin, a component of endothelial cell junctions, causing an increase in transcription of the downstream target gene C-MYC. Importantly, blockade of gap junction intercellular communication prevented β-catenin nuclear translocation. Our findings indicate that maintenance of cell-cell communication is necessary to facilitate a cascade of events that lead to tumor cell migration through the lymphatic endothelium. © 2015 American Heart Association, Inc.

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Growth Associated Protein 43 (GAP-43) as a Novel Target for the Diagnosis, Treatment and Prevention of Epileptogenesis.

PubMed

Nemes, Ashley D; Ayasoufi, Katayoun; Ying, Zhong; Zhou, Qi-Gang; Suh, Hoonkyo; Najm, Imad M

2017-12-18

We previously showed increased growth associated protein 43 (GAP-43) expression in brain samples resected from patients with cortical dysplasia (CD), which was correlated with duration of epilepsy. Here, we used a rat model of CD to examine the regulation of GAP-43 in the brain and serum over the course of epileptogenesis. Baseline GAP-43 expression was higher in CD animals compared to control non-CD rats. An acute seizure increased GAP-43 expression in both CD and control rats. However, GAP-43 expression decreased by day 15 post-seizure in control rats, which did not develop spontaneous seizures. In contrast, GAP-43 remained up-regulated in CD rats, and over 50% developed chronic epilepsy with increased GAP-43 levels in their serum. GAP-43 protein was primarily located in excitatory neurons, suggesting its functional significance in epileptogenesis. Inhibition of GAP-43 expression by shRNA significantly reduced seizure duration and severity in CD rats after acute seizures with subsequent reduction in interictal spiking. Serum GAP-43 levels were significantly higher in CD rats that developed spontaneous seizures. Together, these results suggest GAP-43 as a key factor promoting epileptogenesis, a possible therapeutic target for treatment of progressive epilepsy and a potential biomarker for epilepsy progression in CD.

Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.

PubMed

Song, Baifen; Yang, Xijing; Sun, Hunan; Yu, Liquan; Ma, Jinzhu; Wu, Zhijun; Cui, Yudong

2017-04-01

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC 1-150 ) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC 1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC 1-150 . To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC1 1-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC 1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC 1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC 1-150 . The sera of the immunized mice were collected and the anti-GapC 1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC 1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC 1-150 on the E. coli surface as a vaccine against Streptococcus species. Copyright © 2017. Published by Elsevier Ltd.

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions.

PubMed

El-Mogharbel, Nisrine; Wakefield, Matthew; Deakin, Janine E; Tsend-Ayush, Enkhjargal; Grützner, Frank; Alsop, Amber; Ezaz, Tariq; Marshall Graves, Jennifer A

2007-01-01

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system.

PubMed

Ohtani-Kaneko, Rsituko; Sato, Kenjiro; Tsutiya, Atsuhiro; Nakagawa, Yuka; Hashizume, Kazutoshi; Tazawa, Hidekatsu

2017-10-09

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Global transcriptional analysis of psoriatic skin and blood confirms known disease-associated pathways and highlights novel genomic "hot spots" for differentially expressed genes.

PubMed

Coda, Alvin B; Icen, Murat; Smith, Jason R; Sinha, Animesh A

2012-07-01

There are major gaps in our knowledge regarding the exact mechanisms and genetic basis of psoriasis. To investigate the pathogenesis of psoriasis, gene expression in 10 skin (5 lesional, 5 nonlesional) and 11 blood (6 psoriatic, 5 nonpsoriatic) samples were examined using Affymetrix HG-U95A microarrays. We detected 535 (425 upregulated, 110 downregulated) DEGs in lesional skin at 1% false discovery rate (FDR). Combining nine microarray studies comparing lesional and nonlesional psoriatic skin, 34.5% of dysregulated genes were overlapped in multiple studies. We further identified 20 skin and 2 blood associated transcriptional "hot spots" at specified genomic locations. At 5% FDR, 11.8% skin and 10.4% blood DEGs in our study mapped to one of the 12 PSORS loci. DEGs that overlap with PSORS loci may offer prioritized targets for downstream genetic fine mapping studies. Novel DEG "hot spots" may provide new targets for defining susceptibility loci in future studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

PubMed Central

Struffi, Paolo; Corado, Maria; Kaplan, Leah; Yu, Danyang; Rushlow, Christine; Small, Stephen

2011-01-01

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo. PMID:21865322

Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris.

PubMed

Hobl, Birgit; Hock, Björn; Schneck, Sandra; Fischer, Reinhard; Mack, Matthias

2013-11-01

A novel Pichia pastoris expression vector (pEZT7) for the production of recombinant proteins employing prokaryotic bacteriophage T7 RNA polymerase (T7 RNAP) (EC 2.7.7.6) and the corresponding promoter pT7 was constructed. The gene for T7 RNAP was stably introduced into the P. pastoris chromosome 2 under control of the (endogenous) constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (pGAP). The gene product T7 RNAP was engineered to contain a nuclear localization signal, which directed recombinant T7 RNAP to the P. pastoris nucleus. To promote translation of uncapped T7 RNAP derived transcripts, the internal ribosomal entry site from hepatitis C virus (HCV-IRES) was inserted directly upstream of the multiple cloning site of pEZT7. A P. pastoris autonomous replicating sequence (PARS1) was integrated into pEZT7 enabling propagation and recovery of plasmids from P. pastoris. Rapid amplification of 5' complementary DNA ends (5' RACE) experiments employing the test plasmid pEZT7-EGFP revealed that transcripts indeed initiated at pT7. HCV-IRES mediated translation of the latter mRNAs, however, was not observed. Surprisingly, HCV-IRES and the reverse complement of PARS1 (PARS1rc) were both found to display significant promoter activity as shown by 5' RACE. Copyright © 2013 Elsevier Inc. All rights reserved.

Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

PubMed

Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

2016-11-01

The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

PubMed

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

The distribution and functional properties of Pelizaeus-Merzbacher-like disease-linked Cx47 mutations on Cx47/Cx47 homotypic and Cx47/Cx43 heterotypic gap junctions.

PubMed

Kim, Mi Seong; Gloor, Gregory B; Bai, Donglin

2013-06-01

GJs (gap junctions) allow direct intercellular communication, and consist of Cxs (connexins). In the mammalian central nervous system, oligodendrocytes express Cx47, Cx32 and Cx29, whereas astrocytes express Cx43, Cx30 and Cx26. Homotypic Cx47/Cx47 GJs couple oligodendrocytes, and heterotypic Cx47/Cx43 channels are the primary GJs at oligodendrocyte/astrocyte junctions. Interestingly, autosomal recessive mutations in the gene GJC2 encoding Cx47 have been linked to a central hypomyelinating disease termed PMLD (Pelizaeus-Merzbacher-like disease). The aim of the present study was to determine the cellular distribution and functional properties of PMLD-associated Cx47 mutants (I46M, G149S, G236R, G236S, M286T and T398I). Expressing GFP (green fluorescent protein)-tagged mutant versions of Cx47 in gap-junction-deficient model cells revealed that these mutants were detected at the cell-cell interface similar to that observed for wild-type Cx47. Furthermore, four of the six mutants showed no electrical coupling in both Cx47/Cx47 and Cx47/Cx43 GJ channels. These results suggest that most of the PMLD-linked Cx47 mutants disrupt Cx47/Cx47 and Cx47/Cx43 GJ function in the glial network, which may play a role in leading to PMLD symptoms.

The RasGAP Proteins Ira2 and Neurofibromin Are Negatively Regulated by Gpb1 in Yeast and ETEA in Humans▿

PubMed Central

Phan, Vernon T.; Ding, Vivianne W.; Li, Fenglei; Chalkley, Robert J.; Burlingame, Alma; McCormick, Frank

2010-01-01

The neurofibromatosis type 1 (NF1) gene encodes the GTPase-activating protein (GAP) neurofibromin, which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and neurofibromin (in humans). PMID:20160012

Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment

PubMed Central

Corley, Michael J.; Dye, Christian; D’Antoni, Michelle L.; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M.; Ndhlovu, Lishomwa C.; Maunakea, Alika K.

2016-01-01

Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation “signature” potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI. PMID:27629381

Characterization of the genome and transcriptome of the blue tit Cyanistes caeruleus: polymorphisms, sex-biased expression and selection signals.

PubMed

Mueller, Jakob C; Kuhl, Heiner; Timmermann, Bernd; Kempenaers, Bart

2016-03-01

Decoding genomic sequences and determining their variation within populations has potential to reveal adaptive processes and unravel the genetic basis of ecologically relevant trait variation within a species. The blue tit Cyanistes caeruleus--a long-time ecological model species--has been used to investigate fitness consequences of variation in mating and reproductive behaviour. However, very little is known about the underlying genetic changes due to natural and sexual selection in the genome of this songbird. As a step to bridge this gap, we assembled the first draft genome of a single blue tit, mapped the transcriptome of five females and five males to this reference, identified genomewide variants and performed sex-differential expression analysis in the gonads, brain and other tissues. In the gonads, we found a high number of sex-biased genes, and of those, a similar proportion were sex-limited (genes only expressed in one sex) in males and females. However, in the brain, the proportion of female-limited genes within the female-biased gene category (82%) was substantially higher than the proportion of male-limited genes within the male-biased category (6%). This suggests a predominant on-off switching mechanism for the female-limited genes. In addition, most male-biased genes were located on the Z-chromosome, indicating incomplete dosage compensation for the male-biased genes. We called more than 500,000 SNPs from the RNA-seq data. Heterozygote detection in the single reference individual was highly congruent between DNA-seq and RNA-seq calling. Using information from these polymorphisms, we identified potential selection signals in the genome. We list candidate genes which can be used for further sequencing and detailed selection studies, including genes potentially related to meiotic drive evolution. A public genome browser of the blue tit with the described information is available at http://public-genomes-ngs.molgen.mpg.de. © 2015 John Wiley & Sons Ltd.

Functional characterization of an oxytocin receptor gene variant (rs2268498) previously associated with social cognition by expression analysis in vitro and in human brain biopsy.

PubMed

Reuter, Martin; Montag, Christian; Altmann, Steffen; Bendlow, Fabian; Elger, Christian; Kirsch, Peter; Becker, Albert; Schoch-McGovern, Susanne; Simon, Matthias; Weber, Bernd; Felten, Andrea

2017-10-01

The oxytocin system plays a prominent role in social behavior across species, and numerous genetic studies in humans have reported associations between polymorphisms on the oxytocin receptor (OXTR) gene and phenotypes related to social cognition, affiliation, perspective taking, and sociability in healthy subjects and in patients with atypical social behavior, such as in autism spectrum disorders (ASD). Recently, the first study demonstrating altered agonist-induced OXTR internalization and recycling for the exonic variant rs35062132 emerged. Beside this, there has been no further demonstration of the functionality of the OXTR variants especially there does not exist any for the regulatory units. To address this gap in the literature, we tested the functionality of the promoter flanking single nucleotide polymorphism (SNP) rs2268498, which has proven an interesting candidate for predicting social behavior in recent association studies. Results of genetic expression analyses in human hippocampal tissue showed a twofold difference in messenger RNA transcription, dependent on the presence or absence of the C-allele. This finding was corroborated by cloning, i.e., in vitro reporter gene expression analysis after transfection of OXTR promoter plasmids into HEK-293 cells. Our results underline the importance of OXTR rs2268498 for genetic research in social behavior and ASD.

Evolution and inheritance of early embryonic patterning in D. simulans and D. sechellia

PubMed Central

Lott, Susan E.; Ludwig, Michael Z.; Kreitman, Martin

2010-01-01

Pattern formation in Drosophila is a widely studied example of a robust developmental system. Such robust systems pose a challenge to adaptive evolution, as they mask variation which selection may otherwise act upon. Yet we find variation in the localization of expression domains (henceforth ‘stripe allometry’) in the pattern formation pathway. Specifically, we characterize differences in the gap genes giant and Kruppel, and the pair-rule gene even-skipped, which differ between the sibling species D. simulans and D. sechellia. In a double-backcross experiment, stripe allometry is consistent with maternal inheritance of stripe positioning and multiple genetic factors, with a distinct genetic basis from embryo length. Embryos produced by F1 and F2 backcross mothers exhibit novel spatial patterns of gene expression relative to the parental species, with no measurable increase in positional variance among individuals. Buffering of novel spatial patterns in the backcross genotypes suggests that robustness need not be disrupted in order for the trait to evolve, and perhaps the system is incapable of evolving to prevent the expression of all genetic variation. This limitation, and the ability of natural selection to act on minute genetic differences that are within the “margin of error” for the buffering mechanism, indicates that developmentally buffered traits can evolve without disruption of robustness PMID:21121913

-A curated transcriptomic dataset collection relevant to embryonic development associated with in vitro fertilization in healthy individuals and patients with polycystic ovary syndrome.

PubMed

Mackeh, Rafah; Boughorbel, Sabri; Chaussabel, Damien; Kino, Tomoshige

2017-01-01

The collection of large-scale datasets available in public repositories is rapidly growing and providing opportunities to identify and fill gaps in different fields of biomedical research. However, users of these datasets should be able to selectively browse datasets related to their field of interest. Here we made available a collection of transcriptome datasets related to human follicular cells from normal individuals or patients with polycystic ovary syndrome, in the process of their development, during in vitro fertilization. After RNA-seq dataset exclusion and careful selection based on study description and sample information, 12 datasets, encompassing a total of 85 unique transcriptome profiles, were identified in NCBI Gene Expression Omnibus and uploaded to the Gene Expression Browser (GXB), a web application specifically designed for interactive query and visualization of integrated large-scale data. Once annotated in GXB, multiple sample grouping has been made in order to create rank lists to allow easy data interpretation and comparison. The GXB tool also allows the users to browse a single gene across multiple projects to evaluate its expression profiles in multiple biological systems/conditions in a web-based customized graphical views. The curated dataset is accessible at the following link: http://ivf.gxbsidra.org/dm3/landing.gsp.

Melatonin, Noncoding RNAs, Messenger RNA Stability and Epigenetics—Evidence, Hints, Gaps and Perspectives

PubMed Central

Hardeland, Rüdiger

2014-01-01

Melatonin is a highly pleiotropic regulator molecule, which influences numerous functions in almost every organ and, thus, up- or down-regulates many genes, frequently in a circadian manner. Our understanding of the mechanisms controlling gene expression is actually now expanding to a previously unforeseen extent. In addition to classic actions of transcription factors, gene expression is induced, suppressed or modulated by a number of RNAs and proteins, such as miRNAs, lncRNAs, piRNAs, antisense transcripts, deadenylases, DNA methyltransferases, histone methylation complexes, histone demethylases, histone acetyltransferases and histone deacetylases. Direct or indirect evidence for involvement of melatonin in this network of players has originated in different fields, including studies on central and peripheral circadian oscillators, shift work, cancer, inflammation, oxidative stress, aging, energy expenditure/obesity, diabetes type 2, neuropsychiatric disorders, and neurogenesis. Some of the novel modulators have also been shown to participate in the control of melatonin biosynthesis and melatonin receptor expression. Future work will need to augment the body of evidence on direct epigenetic actions of melatonin and to systematically investigate its role within the network of oscillating epigenetic factors. Moreover, it will be necessary to discriminate between effects observed under conditions of well-operating and deregulated circadian clocks, and to explore the possibilities of correcting epigenetic malprogramming by melatonin. PMID:25310649

­A curated transcriptomic dataset collection relevant to embryonic development associated with in vitro fertilization in healthy individuals and patients with polycystic ovary syndrome

PubMed Central

Mackeh, Rafah; Boughorbel, Sabri; Chaussabel, Damien; Kino, Tomoshige

2017-01-01

The collection of large-scale datasets available in public repositories is rapidly growing and providing opportunities to identify and fill gaps in different fields of biomedical research. However, users of these datasets should be able to selectively browse datasets related to their field of interest. Here we made available a collection of transcriptome datasets related to human follicular cells from normal individuals or patients with polycystic ovary syndrome, in the process of their development, during in vitro fertilization. After RNA-seq dataset exclusion and careful selection based on study description and sample information, 12 datasets, encompassing a total of 85 unique transcriptome profiles, were identified in NCBI Gene Expression Omnibus and uploaded to the Gene Expression Browser (GXB), a web application specifically designed for interactive query and visualization of integrated large-scale data. Once annotated in GXB, multiple sample grouping has been made in order to create rank lists to allow easy data interpretation and comparison. The GXB tool also allows the users to browse a single gene across multiple projects to evaluate its expression profiles in multiple biological systems/conditions in a web-based customized graphical views. The curated dataset is accessible at the following link: http://ivf.gxbsidra.org/dm3/landing.gsp. PMID:28413616

Light-promoted rhodopsin expression and starvation survival in the marine dinoflagellate Oxyrrhis marina.

PubMed

Guo, Zhiling; Zhang, Huan; Lin, Senjie

2014-01-01

The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Genome-wide analysis identifies colonic genes differentially associated with serum leptin and insulin concentrations in C57BL/6J mice fed a high-fat diet.

PubMed

Kim, Sung-Eun; Choo, Jinsil; Yoon, Joon; Chu, Jae Ryang; Bae, Yun Jung; Lee, Seungyeoun; Park, Taesung; Sung, Mi-Kyung

2017-01-01

Obesity-induced chronic inflammation is known to increase the risk of ulcerative colitis, Crohn's disease, and colorectal cancer. Accumulating evidence suggests that leptin and insulin are key molecules linking obesity with diseases of the lower intestine. Here, we identified serum phenotype-associated genes in the colon of diet-induced obese mice as early biomarkers of obesity-associated colonic diseases. C57BL/6J mice were fed with either normal diet (ND, 15% of fat calories) or high-fat diet (HFD, 45% of fat calories) for 8 weeks. Serum concentrations of insulin, insulin-like growth factor-1 (IGF-1), leptin, and adiponectin were measured as obesity-related phenotypic markers. Genome-wide gene expression profiles of colon tissue were determined, followed by statistical analyses to detect differentially expressed and serum phenotype-associated genes. HFD-fed mice showed higher serum concentrations of leptin (P < 0.001) and insulin (P < 0.01) than those in the ND group, whereas serum IGF-1 and adiponectin concentrations did not differ between the two dietary groups. Among differentially expressed genes affected by HFD, 135, 128, 110, and 341 genes were associated with serum levels of leptin, insulin, IGF-1, and adiponectin, respectively. We identified 17 leptin-associated genes and 4 insulin-associated genes that inversely responded to HFD and ND. Among these, leptin-associated Peli3 (Pellino E3 ubiquitin protein ligase family member 3), Creb1 (cAMP responsive element binding protein 1), and Enpp2 (ectonucleotide pyrophosphatase/phosphodiesterase 2, autotaxin) and insulin-associated Centg1 (AGAP2, ArfGAP with GTPase domain) are reported to play a role either in obesity or colonic diseases. mRNA expression of these genes was validated by RT-qPCR. Our data suggest Peli3, Creb1, Enpp2, and Centg1 as potential early biomarker candidates for obesity-induced pathophysiological changes in the colon. Future studies verifying the function of these candidates are needed for the prevention, early detection, and treatment of colon diseases.

Genome-wide analysis identifies colonic genes differentially associated with serum leptin and insulin concentrations in C57BL/6J mice fed a high-fat diet

PubMed Central

Yoon, Joon; Chu, Jae Ryang; Bae, Yun Jung; Lee, Seungyeoun; Park, Taesung; Sung, Mi-Kyung

2017-01-01

Obesity-induced chronic inflammation is known to increase the risk of ulcerative colitis, Crohn’s disease, and colorectal cancer. Accumulating evidence suggests that leptin and insulin are key molecules linking obesity with diseases of the lower intestine. Here, we identified serum phenotype-associated genes in the colon of diet-induced obese mice as early biomarkers of obesity-associated colonic diseases. C57BL/6J mice were fed with either normal diet (ND, 15% of fat calories) or high-fat diet (HFD, 45% of fat calories) for 8 weeks. Serum concentrations of insulin, insulin-like growth factor-1 (IGF-1), leptin, and adiponectin were measured as obesity-related phenotypic markers. Genome-wide gene expression profiles of colon tissue were determined, followed by statistical analyses to detect differentially expressed and serum phenotype-associated genes. HFD-fed mice showed higher serum concentrations of leptin (P < 0.001) and insulin (P < 0.01) than those in the ND group, whereas serum IGF-1 and adiponectin concentrations did not differ between the two dietary groups. Among differentially expressed genes affected by HFD, 135, 128, 110, and 341 genes were associated with serum levels of leptin, insulin, IGF-1, and adiponectin, respectively. We identified 17 leptin-associated genes and 4 insulin-associated genes that inversely responded to HFD and ND. Among these, leptin-associated Peli3 (Pellino E3 ubiquitin protein ligase family member 3), Creb1 (cAMP responsive element binding protein 1), and Enpp2 (ectonucleotide pyrophosphatase/phosphodiesterase 2, autotaxin) and insulin-associated Centg1 (AGAP2, ArfGAP with GTPase domain) are reported to play a role either in obesity or colonic diseases. mRNA expression of these genes was validated by RT-qPCR. Our data suggest Peli3, Creb1, Enpp2, and Centg1 as potential early biomarker candidates for obesity-induced pathophysiological changes in the colon. Future studies verifying the function of these candidates are needed for the prevention, early detection, and treatment of colon diseases. PMID:28170448

Innexin-3 forms connexin-like intercellular channels.

PubMed

Landesman, Y; White, T W; Starich, T A; Shaw, J E; Goodenough, D A; Paul, D L

1999-07-01

Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.

Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits.

PubMed

Carriel, Víctor; Garzón, Ingrid; Campos, Antonio; Cornelissen, Maria; Alaminos, Miguel

2017-02-01

Nerve conduits are promising alternatives for repairing nerve gaps; they provide a close microenvironment that supports nerve regeneration. In this sense, histological analysis of axonal growth is a determinant to achieve successful nerve regeneration. To evaluate this process, the most-used immunohistochemical markers are neurofilament (NF), β-III tubulin and, infrequently, GAP-43. However, GAP-43 expression in long-term nerve regeneration models is still poorly understood. In this study we analysed GAP-43 expression and its correlation with NF and S-100, using three tissue-engineering approaches with different regeneration profiles. A 10 mm gap was created in the sciatic nerve of 12 rats and repaired using collagen conduits or collagen conduits filled with fibrin-agarose hydrogels or with hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs). After 12 weeks the conduits were harvested for histological analysis. Our results confirm the long-term expression of GAP-43 in all groups. The expression of GAP-43 and NF was significantly higher in the group with ADMSCs. Interestingly, GAP-43 was observed in immature, newly formed axons and NF in thicker and mature axons. These proteins were not co-expressed, demonstrating their differential expression in newly formed nerve fascicles. Our descriptive and quantitative histological analysis of GAP-43 and NFL allowed us to determine, with high accuracy, the heterogenic population of axons at different stages of maturation in three tissue-engineering approaches. Finally, to perform a complete assessment of axonal regeneration, the quantitative immunohistochemical evaluation of both GAP-43 and NF could be a useful quality control in tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Lack of Change in Markers of Presynaptic Terminal Abundance Alongside Subtle Reductions in Markers of Presynaptic Terminal Plasticity in Prefrontal Cortex of Schizophrenia Patients

PubMed Central

Fung, Samantha J.; Sivagnanasundaram, Sinthuja; Shannon Weickert, Cynthia

2010-01-01

Background Reduced synaptic connectivity in frontal cortex may contribute to schizophrenia symptoms. While altered mRNA and protein expression of various synaptic genes has been found, discrepancies between studies mean a generalisable synaptic pathology in schizophrenia has not been identified. Methods We determined if mRNAs encoding presynaptic proteins enriched in inhibitory [vesicular GABA transporter (VGAT) and complexin 1] and/or excitatory [vesicular glutamate transporter (VGluT1) and complexin 2] terminals are altered in the dorsolateral prefrontal cortex of subjects with schizophrenia (n=37 patients, n=37 controls). We also measured mRNA expression of markers associated with synaptic plasticity/neurite outgrowth [growth associated protein 43 (GAP43) and neuronal navigators 1 and 2 (NAV1 and NAV2)]; and mRNAs of other synaptic-associated proteins previously implicated in schizophrenia: dysbindin and vesicle-associated membrane protein (VAMP1) mRNAs using quantitative RT-PCR. Results No significant changes in complexin 1, VGAT, complexin 2, VGluT1, dysbindin, NAV2, or VAMP1 mRNA expression were found, however we observed reduced expression of mRNAs associated with plasticity/cytoskeletal modification (GAP43 and NAV1) in schizophrenia. Although dysbindin mRNA did not differ in schizophrenia compared to controls, dysbindin mRNA positively correlated with GAP-43 and NAV1 in schizophrenia, but not in controls, suggesting low levels of dysbindin may be linked to reduced plasticity in the disease state. No relationships between three dysbindin genetic polymorphisms previously associated with dysbindin mRNA levels were found. Conclusions A reduction in the plasticity of synaptic terminals supports the hypothesis that reduced modifiability of synaptic terminals may contribute to neuropathology and working memory deficits in schizophrenia. PMID:21145444

Complexity of gap junctions between horizontal cells of the carp retina.

PubMed

Greb, H; Hermann, S; Dirks, P; Ommen, G; Kretschmer, V; Schultz, K; Zoidl, G; Weiler, R; Janssen-Bienhold, U

2017-01-06

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Computational insights of K1444N substitution in GAP-related domain of NF1 gene associated with neurofibromatosis type 1 disease: a molecular modeling and dynamics approach.

PubMed

Agrahari, Ashish Kumar; Muskan, Meghana; George Priya Doss, C; Siva, R; Zayed, Hatem

2018-05-27

The NF1 gene encodes for neurofibromin protein, which is ubiquitously expressed, but most highly in the central nervous system. Non-synonymous SNPs (nsSNPs) in the NF1 gene were found to be associated with Neurofibromatosis Type 1 disease, which is characterized by the growth of tumors along nerves in the skin, brain, and other parts of the body. In this study, we used several in silico predictions tools to analyze 16 nsSNPs in the RAS-GAP domain of neurofibromin, the K1444N (K1423N) mutation was predicted as the most pathogenic. The comparative molecular dynamic simulation (MDS; 50 ns) between the wild type and the K1444N (K1423N) mutant suggested a significant change in the electrostatic potential. In addition, the RMSD, RMSF, Rg, hydrogen bonds, and PCA analysis confirmed the loss of flexibility and increase in compactness of the mutant protein. Further, SASA analysis revealed exchange between hydrophobic and hydrophilic residues from the core of the RAS-GAP domain to the surface of the mutant domain, consistent with the secondary structure analysis that showed significant alteration in the mutant protein conformation. Our data concludes that the K1444N (K1423N) mutant lead to increasing the rigidity and compactness of the protein. This study provides evidence of the benefits of the computational tools in predicting the pathogenicity of genetic mutations and suggests the application of MDS and different in silico prediction tools for variant assessment and classification in genetic clinics.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

PubMed

Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

2009-08-01

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

Deciphering the genetic control of gene expression following Mycobacterium leprae antigen stimulation.

PubMed

Manry, Jérémy; Nédélec, Yohann; Fava, Vinicius M; Cobat, Aurélie; Orlova, Marianna; Thuc, Nguyen Van; Thai, Vu Hong; Laval, Guillaume; Barreiro, Luis B; Schurr, Erwin

2017-08-01

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection.

RNA sequencing enables systematic identification of platelet transcriptomic alterations in NSCLC patients.

PubMed

Zhang, Qun; Hu, Huan; Liu, Hongda; Jin, Jiajia; Zhu, Peiyuan; Wang, Shujun; Shen, Kaikai; Hu, Yangbo; Li, Zhou; Zhan, Ping; Zhu, Suhua; Fan, Hang; Zhang, Jianya; Lv, Tangfeng; Song, Yong

2018-05-29

Platelets are implicated as key players in the metastatic dissemination of tumor cells. Previous evidence demonstrated platelets retained cytoplasmic RNAs with physiologically activity, splicing pre-mRNA to mRNA and translating into functional proteins in response to external stimulation. Recently, platelets gene profile of healthy or diseased individuals were characterized with the help of RNA sequencing (RNA-Seq) in some studies, leading to new insights into the mechanisms underlying disease pathogenesis. In this study, we performed RNA-seq in platelets from 7 healthy individuals and 15 non-small cell lung cancer (NSCLC) patients. Our data revealed a subset of near universal differently expressed gene (DEG) profiles in platelets of metastatic NSCLC compared to healthy individuals, including 626 up-regulated RNAs (mRNAs and ncRNAs) and 1497 down-regulated genes. The significant over-expressed genes showed enrichment in focal adhesion, platelets activation, gap junction and adherens junction pathways. The DEGs also included previously reported tumor-related genes such as PDGFR, VEGF, EGF, etc., verifying the consistence and significance of platelet RNA-Seq in oncology study. We also validated several up-regulated DEGs involved in tumor cell-induced platelet aggregation (TCIPA) and tumorigenesis. Additionally, transcriptomic comparison analyses of NSCLC subgroups were conducted. Between non-metastatic and metastatic NSCLC patients, 526 platelet DEGs were identified with the most altered expression. The outcomes from subgroup analysis between lung adenocarcinoma and lung squamous cell carcinoma demonstrated the diagnostic potential of platelet RNA-Seq on distinguishing tumor histological types. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canton, Rocio F.; Peijnenburg, Ad A.C.M.; Hoogenboom, Ron L.A.P.

2008-09-01

Hexabromoyclododecane (HBCD), used as flame retardant (FR) mainly in textile industry and in polystyrene foam manufacture, has been identified as a contaminant at levels comparable to other brominated FRs (BFRs). HBCD levels in biota are increasing slowly and seem to reflect the local market demand. The toxicological database of HBCD is too limited to perform at present a solid risk assessment, combining data from exposure and effect studies. In order to fill in some gaps, a 28-day HBCD repeated dose study (OECD407) was done in Wistar rats. In the present work liver tissues from these animals were used for genemore » expression profile analysis. Results show clear gender specificity with females having a higher number of regulated genes and therefore being more sensitive to HBCD than males. Several specific pathways were found to be affected by HBCD exposure, like PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol biosynthesis, and phase I and II pathways. These results were corroborated with quantitative RT-PCR analysis. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females. Genes involved in phase I and II metabolism were up-regulated predominantly in males, which could explain the observed lower HBCD hepatic disposition in male rats in this 28-day study. These sex-specific differences in gene expression profiles could also underlie sex-specific differences in toxicity (e.g. decreased thyroid hormone or increased serum cholesterol levels). To our knowledge, this is the fist study that describes the changes in rat hepatic gene profiles caused by this commonly used flame retardant.« less

The role of B7 costimulation in benzene immunotoxicity and its potential association with cancer risk.

PubMed

Sauer, Elisa; Gauer, Bruna; Nascimento, Sabrina; Nardi, Jessica; Göethel, Gabriela; Costa, Bárbara; Correia, Douglas; Matte, Ursula; Charão, Mariele; Arbo, Marcelo; Duschl, Albert; Moro, Angela; Garcia, Solange Cristina

2018-06-05

Benzene is a recognized human carcinogen; however, there are still some gaps in the knowledge regarding the mechanism of toxicity of this organic solvent and potential early biomarkers for the damage caused by it. In a previous study, our research group demonstrated that the adhesion molecules of the immune system (B7.1 and B7.2) could be potential biomarkers in the early detection of immunotoxicity caused by benzene exposure. Therefore, this study was developed to deepen the understanding regarding this important topic, aiming to contribute to the comprehension of the benzene toxicity mechanism mediated by B7.1 and B7.2 and its potential association with the risk of carcinogenicity. B7.1 and B7.2 protein expression in blood monocytes and B7.1 and B7.2 gene expression in PBMCs were evaluated. Additionally, complement C3 and C4 levels in serum were measured, as well as p53 gene expression in PBMCs. Seventy-four gas station workers (GSW group) and 71 non-occupationally exposed subjects (NEG) were evaluated. Our results demonstrated decreased levels of B7.1 and B7.2 protein and gene expression in the GSW group compared to the NEG (n = 71) (p < 0.01). Along the same lines, decreased levels of the complement system were observed in the GSW group (p < 0.01), demonstrating the impairment of this immune system pathway as well. Additionally, a reduction was observed in p53 gene expression in the GSA group (p < 0.01). These alterations were associated with both the benzene exposure biomarker evaluated, urinary trans, trans-muconic acid, and with exposure time (p < 0.05). Moreover, strong correlations were observed between the gene expression of p53 vs. B7.1 (r = 0.830; p < 0.001), p53 vs. B7.2 (r = 0.685; p < 0.001), and B7.1 vs. B7.2 (r = 0.702; p < 0.001). Taken together, these results demonstrate that the immune system co-stimulatory molecule pathway is affected by benzene exposure. Also, the decrease in p53 gene expression, even at low exposure levels, reinforces the carcinogenicity effect of benzene in this pathway. Therefore, our results suggest that the promotion of immune evasion together with a decrease in p53 gene expression may play an important role in the benzene toxicity mechanism. However, further and targeted studies are needed to confirm this proposition. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

NASA Astrophysics Data System (ADS)

Kanani, S.; Pumir, A.; Krinsky, V.

2008-01-01

One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

The relation between language experience and receptive-expressive semantic gaps in bilingual children

PubMed Central

Gibson, Todd A.; Peña, Elizabeth D.; Bedore, Lisa M.

2018-01-01

The purpose of the current study was to explore the influence of language experience on the presence of the receptive-expressive gap. Each of 778 Spanish-English bilingual children screened pre-kindergarten in Utah and Texas were assigned to one of five language experience groups, ranging from functionally monolingual to balanced bilingual. Children’s scores from the language screener semantics subtest administered in both Spanish and English were standardized, and receptive and expressive semantic scores were compared. Children presented with a meaningful gap between receptive and expressive semantic knowledge in English but not Spanish. This gap increased as target-language exposure decreased. Results indicate that current language experience plays a dominant role in influencing the appearance and magnitude of the receptive-expressive gap. PMID:29670456

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

PubMed

Figueiredo, B C; Almazan, G; Ma, Y; Tetzlaff, W; Miller, F D; Cuello, A C

1993-03-01

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

PubMed

Zhang, Ze; Yu, Angen; Lan, Jiangfeng; Zhang, Hua; Hu, Minqiang; Cheng, Jiewei; Zhao, Lijuan; Lin, Li; Wei, Shun

2017-04-01

Streptococcosis due to the bacterium Streptococcus agalactiae (S. agalactiae) has resulted in enormous economic losses in aquaculture worldwide, especially in the tilapia culture industry. Previously, there were limited vaccines that could be employed against streptococcosis in tilapia. This study aimed to develop a vaccine candidate using the glyceraldehyde-phosphate dehydrogenase protein (GapA) of S. agalactiae encoded by the gapA gene. Tilapia were intraperitoneally injected with PBS, PBS + Freund's adjuvant, PBS + Montanide's adjuvant, GapA + Freund's adjuvant, GapA + Montanide's adjuvant, killed S. agalactiae whole cells (WC)+Freund's adjuvant, or killed S. agalactiae whole cells (WC)+ Montanide's adjuvant. They were then challenged with S. agalactiae, and the relative percentage survival (RPS) was monitored 14 days after the challenge. The highest RPSs were observed in the WC groups, with 76.7% in WC + Freund's adjuvant and 74.4% in WC + Montanide's adjuvant groups; these were followed by the GapA groups, with 63.3% in GapA + Freund's adjuvant and 45.6% in GapA + Montanide's adjuvant groups. The RPS of the PBS group was 0%, and those of PBS + Freund's adjuvant and PBS + Montanide's adjuvant groups were 6.7% and 3.3%, respectively. Additionally, the IgM antibody responses elicited in GapA groups and WC groups were significantly higher than those in PBS groups. Furthermore, the expressions of cytokine (IL-1β and TNF-α) mRNAs in the GapA groups and WC groups were significantly higher than those in the PBS groups. Taken together, these results reveal that the GapA protein is a promising vaccine candidate that could be used to prevent streptococcosis in tilapia. Copyright © 2017 Elsevier Ltd. All rights reserved.

CX43 expression, phosphorylation, and distribution in the normal and autoimmune orchitic testis with a look at gap junctions joining germ cell to germ cell.

PubMed

Pelletier, R-Marc; Akpovi, Casimir D; Chen, Li; Day, Robert; Vitale, María L

2011-01-01

Spermatogenesis requires connexin 43 (Cx43).This study examines normal gene transcription, translation, and phosphorylation of Cx43 to define its role on germ cell growth and Sertoli cell's differentiation, and identifies abnormalities arising from spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and a natural model for autoimmunity. Northern blot analysis detected 2.8- and a 3.7-kb Cx43 mRNA bands in seminiferous tubule-enriched fractions. Cx43 mRNA increased in seminiferous tubule-enriched fractions throughout development and then seasonally with the completion of spermatogenesis. Cx43 protein levels increased transiently during the colonization of the tubules by the early-stage spermatocytes. Cx43 phosphorylated (PCx43) and nonphosphorylated (NPCx43) in Ser368 decreased during the periods of completion of meiosis and Sertoli cell differentiation, while Cx43 mRNA remained elevated throughout. PCx43 labeled chiefly the plasma membrane except by stage VII when vesicles were also labeled in Sertoli cells. Vesicles and lysosomes in Sertoli cells and the Golgi apparatus in the round spermatids were NPCx43 positive. A decrease in Cx43 gene expression was matched by a Cx43 protein increase in the early, not the late, phase of AIO. Total Cx43 and PCx43 decreased with the advance of orchitis. The study makes a novel finding of gap junctions connecting germ cells. The data indicate that Cx43 protein expression and phosphorylation in Ser368 are stage-specific events that may locally influence the acquisition of meiotic competence and the Sertoli cell differentiation in normal testis. AIO modifies Cx43 levels, suggesting changes in Cx43-mediated intercommunication and spermatogenic activity in response to cytokines imbalances in Sertoli cells.

The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development.

PubMed

Tallafuss, A; Wilm, T P; Crozatier, M; Pfeffer, P; Wassef, M; Bally-Cuif, L

2001-10-01

Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, we have searched for vertebrate genes related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. We report here the identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) and shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrate that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. Our results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor.

Adenoviral Mediated Gene Transfer of IGF-1 Enhances Wound Healing and Induces Angiogenesis

PubMed Central

Balaji, S.; LeSaint, M.; Bhattacharya, S. S.; Moles, C.; Dhamija, Y.; Kidd, M.; Le, L.D.; King, A.; Shaaban, A.; Crombleholme, T. M.; Bollyky, P.; Keswani, S. G.

2014-01-01

Background Chronic wounds are characterized by a wound healing and neovascularization deficit. Strategies to increase neovascularization can significantly improve chronic wound healing. Insulin like growth factor (IGF-1) is reported to be a keratinocyte mitogen and is believed to induce angiogenesis via a vascular endothelial growth factor (VEGF) dependent pathway. Using a novel ex vivo human dermal wound model and a diabetic impaired wound healing murine model, we hypothesized that adenoviral over expression of IGF-1 (Ad-IGF-1) will enhance wound healing and induce angiogenesis through a VEGF dependent pathway. Methods Ex vivo: 6 mm full thickness punch biopsies were obtained from normal human skin, and 3 mm full thickness wounds were created at the center. Skin explants were maintained at air liquid interface. Db/db murine model: 8 mm full thickness dorsal wounds in diabetic (db/db) mice were created. Treatment groups in both human ex vivo and in vivo db/db wound models include 1×108 PFU of Ad-IGF-1 or Ad-LacZ, and PBS (n=4–5/group). Cytotoxicity (LDH) was quantified at days 3, 5 and 7 for the human ex vivo wound model. Epithelial gap closure (H&E; Trichrome), VEGF expression (ELISA) and capillary density (CD 31+ CAPS/HPF) were analyzed at day 7. Results In the human ex vivo organ culture, the adenoviral vectors did not demonstrate any significant difference in cytotoxicity compared to PBS. Ad-IGF-1 over expression significantly increases basal keratinocyte migration, with no significant effect on epithelial gap closure. There was a significant increase in capillary density in the Ad-IGF-1 wounds. However, there was no effect on VEGF levels in Ad-IGF-1 samples compared to controls. In db/db wounds, Ad-IGF-1 over expression significantly improves epithelial gap closure and granulation tissue with a dense cellular infiltrate compared to controls. Ad-IGF-1 also increases capillary density, again with no significant difference in VEGF levels in the wounds compared to control treatments. Conclusions In two different models, our data demonstrates that adenoviral mediated gene transfer of IGF-1 results in enhanced wound healing and induces angiogenesis via a VEGF-independent pathway. Understanding the underlying mechanisms of IGF-1 effects on angiogenesis may help produce novel therapeutics for chronic wounds or diseases characterized by a deficit in neovascularization. PMID:24725678

Digital gene expression analysis in the gills of Ruditapes philippinarum exposed to short- and long-term exposures of ammonia nitrogen.

PubMed

Cong, Ming; Wu, Huifeng; Cao, Tengfei; Lv, Jiasen; Wang, Qing; Ji, Chenglong; Li, Chenghua; Zhao, Jianmin

2018-01-01

Previous study revealed severe toxic effects of ammonia nitrogen on Ruditapes philippinarum including lysosomal instability, disturbed metabolic profiles, gill tissues with damaged structure, and variation of neurotransmitter concentrations. However, the underlying molecular mechanism was not fully understood yet. In the present study, digital gene expression technology (DGE) was applied to globally screen the key genes and pathways involved in the responses to short- and long-term exposures of ammonia nitrogen. Results of DGE analysis indicated that short-term duration of ammonia exposure affected pathways in Dorso-ventral axis formation, Notch signaling, thyroid hormone signaling and protein processing in endoplasmic reticulum. The long-term exposure led to DEGs significantly enriched in gap junction, immunity, signal and hormone transduction, as well as key substance metabolism pathways. Functional research of significantly changed DEGs suggested that the immunity of R. philippinarum was weakened heavily by toxic effects of ammonia nitrogen, as well as neuro-transduction and metabolism of important substances. Taken together, the present study provides a molecular support for the previous results of the detrimental toxicity of ammonia exposure in R. philippinarum, further work will be performed to investigate the specific genes and their certain functions involved in ammonia toxicity to molluscs. Copyright © 2017 Elsevier B.V. All rights reserved.

Utilizing social media to study information-seeking and ethical issues in gene therapy.

PubMed

Robillard, Julie M; Whiteley, Louise; Johnson, Thomas Wade; Lim, Jonathan; Wasserman, Wyeth W; Illes, Judy

2013-03-04

The field of gene therapy is rapidly evolving, and while hopes of treating disorders of the central nervous system and ethical concerns have been articulated within the academic community, little is known about views and opinions of different stakeholder groups. To address this gap, we utilized social media to investigate the kind of information public users are seeking about gene therapy and the hopes, concerns, and attitudes they express. We conducted a content analysis of questions containing the keywords "gene therapy" from the Q&A site "Yahoo! Answers" for the 5-year period between 2006 and 2010. From the pool of questions retrieved (N=903), we identified those containing at least one theme related to ethics, environment, economics, law, or society (n=173) and then characterized the content of relevant answers (n=399) through emergent coding. The results show that users seek a wide range of information regarding gene therapy, with requests for scientific information and ethical issues at the forefront of enquiry. The question sample reveals high expectations for gene therapy that range from cures for genetic and nongenetic diseases to pre- and postnatal enhancement of physiological attributes. Ethics questions are commonly expressed as fears about the impact of gene therapy on self and society. The answer sample echoes these concerns but further suggests that the acceptability of gene therapy varies depending on the specific application. Overall, the findings highlight the powerful role of social media as a rich resource for research into attitudes toward biomedicine and as a platform for knowledge exchange and public engagement for topics relating to health and disease.

Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome.

PubMed

Chen, Meili; Hu, Yibo; Liu, Jingxing; Wu, Qi; Zhang, Chenglin; Yu, Jun; Xiao, Jingfa; Wei, Fuwen; Wu, Jiayan

2015-12-11

High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

PubMed

Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

2015-11-01

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A method for the further assembly of targeted unigenes in a transcriptome after assembly by Trinity

PubMed Central

Xiao, Xinlong; Ma, Jinbiao; Sun, Yufang; Yao, Yinan

2015-01-01

RNA-sequencing has been widely used to obtain high throughput transcriptome sequences in various species, but the assembly of a full set of complete transcripts is still a significant challenge. Judging by the number of expected transcripts and assembled unigenes in a transcriptome library, we believe that some unigenes could be reassembled. In this study, using the nitrate transporter (NRT) gene family and phosphate transporter (PHT) gene family in Salicornia europaea as examples, we introduced an approach to further assemble unigenes found in transcriptome libraries which had been previously generated by Trinity. To find the unigenes of a particular transcript that contained gaps, we respectively selected 16 NRT candidate unigene pairs and 12 PHT candidate unigene pairs for which the two unigenes had the same annotations, the same expression patterns among various RNA-seq samples, and different positions of the proteins coded as mapped to a reference protein. To fill a gap between the two unigenes, PCR was performed using primers that mapped to the two unigenes and the PCR products were sequenced, which demonstrated that 5 unigene pairs of NRT and 3 unigene pairs of PHT could be reassembled when the gaps were filled using the corresponding PCR product sequences. This fast and simple method will reduce the redundancy of targeted unigenes and allow acquisition of complete coding sequences (CDS). PMID:26528307

Cis-eQTL-based trans-ethnic meta-analysis reveals novel genes associated with breast cancer risk

PubMed Central

Tai, Caroline G.; Passarelli, Michael N.; Hu, Donglei; Huntsman, Scott; Zaitlen, Noah; Ziv, Elad; Witte, John S.

2017-01-01

Breast cancer is the most common solid organ malignancy and the most frequent cause of cancer death among women worldwide. Previous research has yielded insights into its genetic etiology, but there remains a gap in the understanding of genetic factors that contribute to risk, and particularly in the biological mechanisms by which genetic variation modulates risk. The National Cancer Institute’s “Up for a Challenge” (U4C) competition provided an opportunity to further elucidate the genetic basis of the disease. Our group leveraged the seven datasets made available by the U4C organizers and data from the publicly available UK Biobank cohort to examine associations between imputed gene expression and breast cancer risk. In particular, we used reference datasets describing the breast tissue and whole blood transcriptomes to impute expression levels in breast cancer cases and controls. In trans-ethnic meta-analyses of U4C and UK Biobank data, we found significant associations between breast cancer risk and the expression of RCCD1 (joint p-value: 3.6x10-06) and DHODH (p-value: 7.1x10-06) in breast tissue, as well as a suggestive association for ANKLE1 (p-value: 9.3x10-05). Expression of RCCD1 in whole blood was also suggestively associated with disease risk (p-value: 1.2x10-05), as were expression of ACAP1 (p-value: 1.9x10-05) and LRRC25 (p-value: 5.2x10-05). While genome-wide association studies (GWAS) have implicated RCCD1 and ANKLE1 in breast cancer risk, they have not identified the remaining three genes. Among the genetic variants that contributed to the predicted expression of the five genes, we found 23 nominally (p-value < 0.05) associated with breast cancer risk, among which 15 are not in high linkage disequilibrium with risk variants previously identified by GWAS. In summary, we used a transcriptome-based approach to investigate the genetic underpinnings of breast carcinogenesis. This approach provided an avenue for deciphering the functional relevance of genes and genetic variants involved in breast cancer. PMID:28362817

Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative Real-Time PCR.

PubMed

Olioso, Debora; Marzotto, Marta; Bonafini, Clara; Brizzi, Maurizio; Bellavite, Paolo

2016-05-01

Arnica montana is a popular traditional remedy widely used in complementary medicine, also for its wound healing properties. Despite its acknowledged action in clinical settings at various doses, the molecular aspects relating to how A. montana promotes wound healing remain to be elucidated. To fill this gap, we evaluated the whole plant extract, in a wide range of dilutions, in THP-1 human cells, differentiated into mature macrophages and into an alternative IL-4-activated phenotype involved in tissue remodelling and healing. Real-time quantitative Reverse Transcription Polymerase Chain Reaction (PCR) analysis was used to study the changes in the expression of a customized panel of key genes, mainly cytokines, receptors and transcription factors. On macrophages differentiated towards the wound healing phenotype, A. montana affected the expression of several genes. In particular CXC chemokine ligand 1 (CXCL1), coding for an chief chemokine, exhibited the most consistent increase of expression, while also CXC chemokine ligand 2 (CXCL2), Interleukin8 (IL8) and bone morphogenetic protein (BMP2) were slightly up-regulated, suggesting a positive influence of A. montana on neutrophil recruitment and on angiogenesis. MMP1, coding for a metalloproteinase capable of cleaving extracellular matrix substrates, was down-regulated. Most results showed non-linearity of the dose-effect relationship. This exploratory study provides new insights into the cellular and molecular mechanisms of action of A. montana as a promoter of healing, since some of the genes it modifies are key regulators of tissue remodelling, inflammation and chemotaxis. Copyright © 2016 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

ALE: automated label extraction from GEO metadata.

PubMed

Giles, Cory B; Brown, Chase A; Ripperger, Michael; Dennis, Zane; Roopnarinesingh, Xiavan; Porter, Hunter; Perz, Aleksandra; Wren, Jonathan D

2017-12-28

NCBI's Gene Expression Omnibus (GEO) is a rich community resource containing millions of gene expression experiments from human, mouse, rat, and other model organisms. However, information about each experiment (metadata) is in the format of an open-ended, non-standardized textual description provided by the depositor. Thus, classification of experiments for meta-analysis by factors such as gender, age of the sample donor, and tissue of origin is not feasible without assigning labels to the experiments. Automated approaches are preferable for this, primarily because of the size and volume of the data to be processed, but also because it ensures standardization and consistency. While some of these labels can be extracted directly from the textual metadata, many of the data available do not contain explicit text informing the researcher about the age and gender of the subjects with the study. To bridge this gap, machine-learning methods can be trained to use the gene expression patterns associated with the text-derived labels to refine label-prediction confidence. Our analysis shows only 26% of metadata text contains information about gender and 21% about age. In order to ameliorate the lack of available labels for these data sets, we first extract labels from the textual metadata for each GEO RNA dataset and evaluate the performance against a gold standard of manually curated labels. We then use machine-learning methods to predict labels, based upon gene expression of the samples and compare this to the text-based method. Here we present an automated method to extract labels for age, gender, and tissue from textual metadata and GEO data using both a heuristic approach as well as machine learning. We show the two methods together improve accuracy of label assignment to GEO samples.

Growth hormone transgenesis in coho salmon disrupts muscle immune function impacting cross-talk with growth systems.

PubMed

Alzaid, Abdullah; Kim, Jin-Hyoung; Devlin, Robert H; Martin, Samuel A M; Macqueen, Daniel J

2018-04-26

Suppression of growth during infection may aid resource allocation towards effective immune function. Past work supporting this hypothesis in salmonid fish revealed an immune-responsive regulation of the insulin-like growth factor (IGF) system, an endocrine pathway downstream of growth hormone (GH). Skeletal muscle is the main target for growth and energetic storage in fish, yet little is known about how its growth is regulated during an immune response. We addressed this knowledge gap by characterizing muscle immune responses in size-matched coho salmon ( Oncorhynchus kisutch ) achieving different growth rates. We compared a wild-type strain with two GH transgenic groups from the same genetic background achieving either maximal or suppressed growth, a design separating GH's direct effects from its influence on growth rate and nutritional state. Fish were sampled 30h post-injection with PBS (control) or mimics of bacterial or viral infection. We quantified mRNA expression levels for genes from the GH, GH receptor, IGF hormone, IGF1 receptor and IGF-binding protein families, along with immune genes involved in inflammatory or antiviral responses and muscle growth status marker genes. We demonstrate dampened immune function in GH transgenics compared to wild-type. The muscle of GH transgenics achieving rapid growth showed no detectable antiviral response, coupled with evidence of a constitutive inflammatory state. GH and IGF system gene expression was strongly altered by GH transgenesis and fast growth, both for baseline expression and responses to immune stimulation. Thus, GH transgenesis strongly disrupts muscle immune status and normal GH and IGF system expression responses to immune stimulation. © 2018. Published by The Company of Biologists Ltd.

Independent Losses of Visual Perception Genes Gja10 and Rbp3 in Echolocating Bats (Order: Chiroptera)

PubMed Central

Shen, Bin; Fang, Tao; Dai, Mengyao; Jones, Gareth; Zhang, Shuyi

2013-01-01

A trade-off between the sensory modalities of vision and hearing is likely to have occurred in echolocating bats as the sophisticated mechanism of laryngeal echolocation requires considerable neural processing and has reduced the reliance of echolocating bats on vision for perceiving the environment. If such a trade-off exists, it is reasonable to hypothesize that some genes involved in visual function may have undergone relaxed selection or even functional loss in echolocating bats. The Gap junction protein, alpha 10 (Gja10, encoded by Gja10 gene) is expressed abundantly in mammal retinal horizontal cells and plays an important role in horizontal cell coupling. The interphotoreceptor retinoid-binding protein (Irbp, encoded by the Rbp3 gene) is mainly expressed in interphotoreceptor matrix and is known to be critical for normal functioning of the visual cycle. We sequenced Gja10 and Rbp3 genes in a taxonomically wide range of bats with divergent auditory characteristics (35 and 18 species for Gja10 and Rbp3, respectively). Both genes have became pseudogenes in species from the families Hipposideridae and Rhinolophidae that emit constant frequency echolocation calls with Doppler shift compensation at high-duty-cycles (the most sophisticated form of biosonar known), and in some bat species that emit echolocation calls at low-duty-cycles. Our study thus provides further evidence for the hypothesis that a trade-off occurs at the genetic level between vision and echolocation in bats. PMID:23874796

Independent losses of visual perception genes Gja10 and Rbp3 in echolocating bats (Order: Chiroptera).

PubMed

Shen, Bin; Fang, Tao; Dai, Mengyao; Jones, Gareth; Zhang, Shuyi

2013-01-01

A trade-off between the sensory modalities of vision and hearing is likely to have occurred in echolocating bats as the sophisticated mechanism of laryngeal echolocation requires considerable neural processing and has reduced the reliance of echolocating bats on vision for perceiving the environment. If such a trade-off exists, it is reasonable to hypothesize that some genes involved in visual function may have undergone relaxed selection or even functional loss in echolocating bats. The Gap junction protein, alpha 10 (Gja10, encoded by Gja10 gene) is expressed abundantly in mammal retinal horizontal cells and plays an important role in horizontal cell coupling. The interphotoreceptor retinoid-binding protein (Irbp, encoded by the Rbp3 gene) is mainly expressed in interphotoreceptor matrix and is known to be critical for normal functioning of the visual cycle. We sequenced Gja10 and Rbp3 genes in a taxonomically wide range of bats with divergent auditory characteristics (35 and 18 species for Gja10 and Rbp3, respectively). Both genes have became pseudogenes in species from the families Hipposideridae and Rhinolophidae that emit constant frequency echolocation calls with Doppler shift compensation at high-duty-cycles (the most sophisticated form of biosonar known), and in some bat species that emit echolocation calls at low-duty-cycles. Our study thus provides further evidence for the hypothesis that a trade-off occurs at the genetic level between vision and echolocation in bats.

Anti-stress and neuronal cell differentiation induction effects of Rosmarinus officinalis L. essential oil.

PubMed

Villareal, Myra O; Ikeya, Ayumi; Sasaki, Kazunori; Arfa, Abdelkarim Ben; Neffati, Mohamed; Isoda, Hiroko

2017-12-22

Mood disorder accounts for 13 % of global disease burden. And while therapeutic agents are available, usually orally administered, most have unwanted side effects, and thus making the inhalation of essential oils (EOs) an attractive alternative therapy. Rosmarinus officinalis EO (ROEO), Mediterranean ROEO reported to improve cognition, mood, and memory, the effect on stress of which has not yet been determined. Here, the anti-stress effect of ROEO on stress was evaluated in vivo and in vitro. Six-week-old male ICR mice were made to inhale ROEO and subjected to tail suspension test (TST). To determine the neuronal differentiation effect of ROEO in vitro, induction of ROEO-treated PC12 cells differentiation was observed. Intracellular acetylcholine and choline, as well as the Gap43 gene expression levels were also determined. Inhalation of ROEO significantly decreased the immobility time of ICR mice and serum corticosterone level, accompanied by increased brain dopamine level. Determination of the underlying mechanism in vitro revealed a PC12 differentiation-induction effect through the modulation of intracellular acetylcholine, choline, and Gap43 gene expression levels. ROEO activates the stress response system through the NGF pathway and the hypothalamus-pituitary-adrenal axis, promoting dopamine production and secretion. The effect of ROEO may be attributed to its bioactive components, specifically to α-pinene, one of its major compounds that has anxiolytic property. The results of this study suggest that ROEO inhalation has therapeutic potential against stress-related psychiatric disorders.

Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

PubMed

Sirey, Tamara M; Ponting, Chris P

2016-10-15

The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

Analysis of Pacific oyster larval proteome and its response to high-CO2.

PubMed

Dineshram, R; Wong, Kelvin K W; Xiao, Shu; Yu, Ziniu; Qian, Pei Yuan; Thiyagarajan, Vengatesen

2012-10-01

Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO(2) due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO(2). Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

PubMed Central

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

2015-01-01

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

Conifer genomics and adaptation: at the crossroads of genetic diversity and genome function.

PubMed

Prunier, Julien; Verta, Jukka-Pekka; MacKay, John J

2016-01-01

Conifers have been understudied at the genomic level despite their worldwide ecological and economic importance but the situation is rapidly changing with the development of next generation sequencing (NGS) technologies. With NGS, genomics research has simultaneously gained in speed, magnitude and scope. In just a few years, genomes of 20-24 gigabases have been sequenced for several conifers, with several others expected in the near future. Biological insights have resulted from recent sequencing initiatives as well as genetic mapping, gene expression profiling and gene discovery research over nearly two decades. We review the knowledge arising from conifer genomics research emphasizing genome evolution and the genomic basis of adaptation, and outline emerging questions and knowledge gaps. We discuss future directions in three areas with potential inputs from NGS technologies: the evolutionary impacts of adaptation in conifers based on the adaptation-by-speciation model; the contributions of genetic variability of gene expression in adaptation; and the development of a broader understanding of genetic diversity and its impacts on genome function. These research directions promise to sustain research aimed at addressing the emerging challenges of adaptation that face conifer trees. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture.

PubMed

Orlandi, Alessia; Pagani, Francesca; Avitabile, Daniele; Bonanno, Giuseppina; Scambia, Giovanni; Vigna, Elisa; Grassi, Francesca; Eusebi, Fabrizio; Fucile, Sergio; Pesce, Maurizio; Capogrossi, Maurizio C

2008-04-01

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Evolution and inheritance of early embryonic patterning in Drosophila simulans and D. sechellia.

PubMed

Lott, Susan E; Ludwig, Michael Z; Kreitman, Martin

2011-05-01

Pattern formation in Drosophila is a widely studied example of a robust developmental system. Such robust systems pose a challenge to adaptive evolution, as they mask variation that selection may otherwise act upon. Yet we find variation in the localization of expression domains (henceforth "stripe allometry") in the pattern formation pathway. Specifically, we characterize differences in the gap genes giant and Kruppel, and the pair-rule gene even-skipped, which differ between the sibling species Drosophila simulans and D. sechellia. In a double-backcross experiment, stripe allometry is consistent with maternal inheritance of stripe positioning and multiple genetic factors, with a distinct genetic basis from embryo length. Embryos produced by F1 and F2 backcross mothers exhibit novel spatial patterns of gene expression relative to the parental species, with no measurable increase in positional variance among individuals. Buffering of novel spatial patterns in the backcross genotypes suggests that robustness need not be disrupted in order for the trait to evolve, and perhaps the system is incapable of evolving to prevent the expression of all genetic variation. This limitation, and the ability of natural selection to act on minute genetic differences that are within the "margin of error" for the buffering mechanism, indicates that developmentally buffered traits can evolve without disruption of robustness. © 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.

Variations in gap junctional intercellular communication and connexin expression in fibroblasts derived from keloid and hypertrophic scars.

PubMed

Lu, Feng; Gao, JianHua; Ogawa, Rei; Hyakusoku, Hiko

2007-03-01

Expression of connexins and other constituent proteins of gap junctions along with gap junctional intercellular communication are involved in cellular development and differentiation processes. In addition, an increasing number of hereditary skin disorders appear to be linked to connexins. Therefore, in this report, the authors studied in vitro gap junctional intercellular communication function and connexin expression in fibroblasts derived from keloid and hypertrophic scar patients. Fibroblasts harvested from each of six keloid and hypertrophic scar patients were used for this study. Gap junctional intercellular communication function was investigated using the gap fluorescence recovery after photobleaching method, and expression of connexin proteins was studied using quantitative confocal microscopic analyses. Compared with normal skin, a decreased level of gap junctional intercellular communication was seen in fibroblasts derived from hypertrophic scar tissue, whereas an extremely low gap junctional intercellular communication level was detected in fibroblasts derived from keloid tissue. We also detected little connexin 43 (Cx43) protein localized in fibroblasts derived from keloids. Moreover, Cx43 protein levels were much lower in fibroblasts derived from hypertrophic scars than in those derived from normal skin. The authors' data suggest that the loss of gap junctional intercellular communication and connexin expression may affect intercellular recognition and thus break the proliferation and apoptosis balance in fibroblasts derived from keloid and hypertrophic scar tissue.

Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation.

PubMed

Wan, Xiaojuan; Wang, Songbo; Xu, Jingren; Zhuang, Lu; Xing, Kongping; Zhang, Mengyuan; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Sun, Jiajie; Zhang, Yongliang; Li, Tiejun; Shu, Gang; Jiang, Qingyan

2017-01-01

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

A Gap in Time: Extending our Knowledge of Temporal Processing Deficits in the HIV-1 Transgenic Rat.

PubMed

McLaurin, Kristen A; Moran, Landhing M; Li, Hailong; Booze, Rosemarie M; Mactutus, Charles F

2017-03-01

Approximately 50 % of HIV-1 seropositive individuals develop HIV-1 associated neurocognitive disorders (HAND), which commonly include alterations in executive functions, such as inhibition, set shifting, and complex problem solving. Executive function deficits in HIV-1 are fairly well characterized, however, relatively few studies have explored the elemental dimensions of neurocognitive impairment in HIV-1. Deficits in temporal processing, caused by HIV-1, may underlie the symptoms of impairment in higher level cognitive processes. Translational measures of temporal processing, including cross-modal prepulse inhibition (PPI), gap-prepulse inhibition (gap-PPI), and gap threshold detection, were studied in mature (ovariectomized) female HIV-1 transgenic (Tg) rats, which express 7 of the 9 HIV-1 genes constitutively throughout development. Cross-modal PPI revealed a relative insensitivity to the manipulation of interstimulus interval (ISI) in HIV-1 Tg animals in comparison to control animals, extending previously reported temporal processing deficits in HIV-1 Tg rats to a more advanced age, suggesting the permanence of temporal processing deficits. In gap-PPI, HIV-1 Tg animals exhibited a relative insensitivity to the manipulation of ISI in comparison to control animals. In gap-threshold detection, HIV-1 Tg animals displayed a profound differential sensitivity to the manipulation of gap duration. Presence of the HIV-1 transgene was diagnosed with 91.1 % accuracy using gap threshold detection measures. Understanding the generality and permanence of temporal processing deficits in the HIV-1 Tg rat is vital to modeling neurocognitive deficits observed in HAND and provides a key target for the development of a diagnostic screening tool.

ADAGE signature analysis: differential expression analysis with data-defined gene sets.

PubMed

Tan, Jie; Huyck, Matthew; Hu, Dongbo; Zelaya, René A; Hogan, Deborah A; Greene, Casey S

2017-11-22

Gene set enrichment analysis and overrepresentation analyses are commonly used methods to determine the biological processes affected by a differential expression experiment. This approach requires biologically relevant gene sets, which are currently curated manually, limiting their availability and accuracy in many organisms without extensively curated resources. New feature learning approaches can now be paired with existing data collections to directly extract functional gene sets from big data. Here we introduce a method to identify perturbed processes. In contrast with methods that use curated gene sets, this approach uses signatures extracted from public expression data. We first extract expression signatures from public data using ADAGE, a neural network-based feature extraction approach. We next identify signatures that are differentially active under a given treatment. Our results demonstrate that these signatures represent biological processes that are perturbed by the experiment. Because these signatures are directly learned from data without supervision, they can identify uncurated or novel biological processes. We implemented ADAGE signature analysis for the bacterial pathogen Pseudomonas aeruginosa. For the convenience of different user groups, we implemented both an R package (ADAGEpath) and a web server ( http://adage.greenelab.com ) to run these analyses. Both are open-source to allow easy expansion to other organisms or signature generation methods. We applied ADAGE signature analysis to an example dataset in which wild-type and ∆anr mutant cells were grown as biofilms on the Cystic Fibrosis genotype bronchial epithelial cells. We mapped active signatures in the dataset to KEGG pathways and compared with pathways identified using GSEA. The two approaches generally return consistent results; however, ADAGE signature analysis also identified a signature that revealed the molecularly supported link between the MexT regulon and Anr. We designed ADAGE signature analysis to perform gene set analysis using data-defined functional gene signatures. This approach addresses an important gap for biologists studying non-traditional model organisms and those without extensive curated resources available. We built both an R package and web server to provide ADAGE signature analysis to the community.

Comparing the transcriptomes of embryos from domesticated and wild Atlantic salmon (Salmo salar L.) stocks and examining factors that influence heritability of gene expression.

PubMed

Bicskei, Beatrix; Taggart, John B; Glover, Kevin A; Bron, James E

2016-03-17

Due to selective breeding, domesticated and wild Atlantic salmon are genetically diverged, which raises concerns about farmed escapees having the potential to alter the genetic composition of wild populations and thereby disrupting local adaptation. Documenting transcriptional differences between wild and domesticated stocks under controlled conditions is one way to explore the consequences of domestication and selection. We compared the transcriptomes of wild and domesticated Atlantic salmon embryos, by using a custom 44k oligonucleotide microarray to identify perturbed gene pathways between the two stocks, and to document the inheritance patterns of differentially-expressed genes by examining gene expression in their reciprocal hybrids. Data from 24 array interrogations were analysed: four reciprocal cross types (W♀ × W♂, D♀ × W♂; W♀ × D♂, D♀ × D♂) × six biological replicates. A common set of 31,491 features on the microarrays passed quality control, of which about 62 % were assigned a KEGG Orthology number. A total of 6037 distinct genes were identified for gene-set enrichment/pathway analysis. The most highly enriched functional groups that were perturbed between the two stocks were cellular signalling and immune system, ribosome and RNA transport, and focal adhesion and gap junction pathways, relating to cell communication and cell adhesion molecules. Most transcripts that were differentially expressed between the stocks were governed by additive gene interaction (33 to 42 %). Maternal dominance and over-dominance were also prevalent modes of inheritance, with no convincing evidence for a stock effect. Our data indicate that even at this relatively early developmental stage, transcriptional differences exist between the two stocks and affect pathways that are relevant to wild versus domesticated environments. Many of the identified differentially perturbed pathways are involved in organogenesis, which is expected to be an active process at the eyed egg stage. The dominant effects are more largely due to the maternal line than to the origin of the stock. This finding is particularly relevant in the context of potential introgression between farmed and wild fish, since female escapees tend to have a higher spawning success rate compared to males.

The expression dynamics of mechanosensitive genes in extra-embryonic vasculature after heart starts to beat in chick embryo.

PubMed

Rajendran, Saranya; Sundaresan, Lakshmikirupa; Rajendran, Krithika; Selvaraj, Monica; Gupta, Ravi; Chatterjee, Suvro

2016-02-11

Fluid flow plays an important role in vascular development. However, the detailed mechanisms, particularly the link between flow and modulation of gene expression during vascular development, remain unexplored. In chick embryo, the key events of vascular development from initiation of heart beat to establishment of effective blood flow occur between the stages HH10 and HH13. Therefore, we propose a novel in vivo model to study the flow experienced by developing endothelium. Using this model, we aimed to capture the transcriptome dynamics of the pre- and post-flow conditions. RNA was isolated from extra embryonic area vasculosa (EE-AV) pooled from three chick embryos between HH10-HH13 and RNA sequencing was performed. The whole transcriptome sequencing of chick identified up-regulation of some of the previously well-known mechanosensitive genes including NFR2, HAND1, CTGF and KDR. GO analyses of the up-regulated genes revealed enrichment of several biological processes including heart development, extracellular matrix organization, cell-matrix adhesion, cell migration, blood vessel development, patterning of blood vessels, collagen fibril organization. Genes encoding for gap junctions proteins which are involved in vascular remodeling and arterial-venous differentiation, and genes involved in cell-cell adhesion, and ECM interactions were significantly up-regulated. Validation of selected genes through semi quantitative PCR was performed. The study indicates that shear stress plays a major role in development. Through appropriate validation, this platform can serve as an in vivo model to study conditions of disturbed flow in pathology as well as normal flow during development.

Mechanisms of transgenerational inheritance of addictive-like behaviors.

PubMed

Vassoler, F M; Sadri-Vakili, G

2014-04-04

Genetic factors are implicated in the heritability of drug abuse. However, even with advances in current technology no specific genes have been identified that are critical for the transmission of drug-induced phenotypes to subsequent generations. It is now evident that epigenetic factors contribute to disease heritability and represent a link between genes and the environment. Recently, epigenetic mechanisms have been shown to underlie drug-induced structural, synaptic, and behavioral plasticity by coordinating the expression of gene networks within the brain. Therefore, the epigenome provides a direct mechanism for drugs of abuse to influence the genetic events involved in the development of addiction as well as its heritability to subsequent generations. In this review we discuss the mechanisms underlying intergenerational epigenetic transmission, highlight studies that demonstrate this phenomenon with particular attention to the field of addiction, and identify gaps for future studies. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas.

PubMed

Alaga, Katanya C; Crawford, Melissa; Dagnino, Lina; Laird, Dale W

2017-01-01

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas

PubMed Central

Alaga, Katanya C.; Crawford, Melissa; Dagnino, Lina; Laird, Dale W.

2017-01-01

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures. PMID:28607585

Complete genome sequencing of the luminescent bacterium, Vibrio qinghaiensis sp. Q67 using PacBio technology

NASA Astrophysics Data System (ADS)

Gong, Liang; Wu, Yu; Jian, Qijie; Yin, Chunxiao; Li, Taotao; Gupta, Vijai Kumar; Duan, Xuewu; Jiang, Yueming

2018-01-01

Vibrio qinghaiensis sp.-Q67 (Vqin-Q67) is a freshwater luminescent bacterium that continuously emits blue-green light (485 nm). The bacterium has been widely used for detecting toxic contaminants. Here, we report the complete genome sequence of Vqin-Q67, obtained using third-generation PacBio sequencing technology. Continuous long reads were attained from three PacBio sequencing runs and reads >500 bp with a quality value of >0.75 were merged together into a single dataset. This resultant highly-contiguous de novo assembly has no genome gaps, and comprises two chromosomes with substantial genetic information, including protein-coding genes, non-coding RNA, transposon and gene islands. Our dataset can be useful as a comparative genome for evolution and speciation studies, as well as for the analysis of protein-coding gene families, the pathogenicity of different Vibrio species in fish, the evolution of non-coding RNA and transposon, and the regulation of gene expression in relation to the bioluminescence of Vqin-Q67.

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

PubMed

Wang, Meng; Li, Sijin; Zhao, Huimin

2016-01-01

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

PubMed Central

2014-01-01

Background Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms. PMID:24885240

mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice.

PubMed

Boyer, Alexandre; Girard, Meggie; Thimmanahalli, Dayananda S; Levasseur, Adrien; Céleste, Christophe; Paquet, Marilène; Duggavathi, Rajesha; Boerboom, Derek

2016-07-01

The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood-testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtor(flox/flox); Amhr2(cre/+) mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtor(flox/flox);Amhr2(cre/+) mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtor(flox/flox); Amhr2(cre/+) mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage. © 2016 by the Society for the Study of Reproduction, Inc.

Integrative genetic analysis of transcription modules: towards filling the gap between genetic lociand inherited traits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Hongqiang; Chen, Hao; Bao, Lei

2005-01-01

Genetic loci that regulate inherited traits are routinely identified using quantitative trait locus (QTL) mapping methods. However, the genotype-phenotype associations do not provide information on the gene expression program through which the genetic loci regulate the traits. Transcription modules are 'selfconsistent regulatory units' and are closely related to the modular components of gene regulatory network [Ihmels, J., Friedlander, G., Bergmann, S., Sarig, O., Ziv, Y. and Barkai, N. (2002) Revealing modular organization in the yeast transcriptional network. Nat. Genet., 31, 370-377; Segal, E., Shapira, M., Regev, A., Pe'er, D., Botstein, D., Koller, D. and Friedman, N. (2003) Module networks: identifyingmore » regulatory modules and their condition-specific regulators from gene expression data. Nat. Genet., 34, 166-176]. We used genome-wide genotype and gene expression data of a genetic reference population that consists of mice of 32 recombinant inbred strains to identify the transcription modules and the genetic loci regulating them. Twenty-nine transcription modules defined by genetic variations were identified. Statistically significant associations between the transcription modules and 18 classical physiological and behavioral traits were found. Genome-wide interval mapping showed that major QTLs regulating the transcription modules are often co-localized with the QTLs regulating the associated classical traits. The association and the possible co-regulation of the classical trait and transcription module indicate that the transcription module may be involved in the gene pathways connecting the QTL and the classical trait. Our results show that a transcription module may associate with multiple seemingly unrelated classical traits and a classical trait may associate with different modules. Literature mining results provided strong independent evidences for the relations among genes of the transcription modules, genes in the regions of the QTLs regulating the transcription modules and the keywords representing the classical traits.« less

Research Report: Intermittent hypobaric hypoxia and hyperbaric oxygen on GAP-43 in the rat carotid body.

PubMed

Peng, Zhengwu; Fan, Juan; Liu, Ling; Kuang, Fang; Xue, Fen; Wang, Bairen

2015-01-01

Adaptive changes in the carotid body (CB) including the expression of the growth-associated protein-43 (GAP-43) have been studied in response to low, but not high, oxygen exposure. Expression of GAP-43 in the CB of rats under different atmospheric pressures and oxygen partial pressure (PO2) conditions was investigated. Mature male Sprague-Dawley rats were exposed to intermittent hypobaric hypoxia (IHH, 0, 1, 2 and 3 weeks), intermittent hyperbaric oxygen (IHBO2, 0, 1, 5 and 10 days, sacrificed six hours or 24 hours after the last HBO2 exposure), and intermittent hyperbaric normoxia (IHN, same treatment pattern as IHBO2). GAP-43 was highly expressed (mainly in type I cells) in the CB of normal rats. IHH u-regulated GAP-43 expression in the CB with significant differences (immunohistochemical staining [IHC]: F(3,15)=40.64, P < 0.01; western blot [WB]: F(3,16) = 53.52, P < 0.01) across the subgroups. GAP-43 expression in the CB was inhibited by IHBO2 (controls vs. IHBO2 groups, IHC: F(6,30) = 15.85, P < 0.01; WB: F(6,29) = 15.95, P < 0.01). No detectable changes in GAP-43 expression were found for IHN. These findings indicated that different PO2 conditions, but not air pressures, played an important role in the plasticity of the CB, and that GAP-43 might be a viable factor for the plasticity of the CB.

Mammalian enabled (Mena) is a critical regulator of cardiac function

PubMed Central

Aguilar, Frédérick; Belmonte, Stephen L.; Ram, Rashmi; Noujaim, Sami F.; Dunaevsky, Olga; Protack, Tricia L.; Jalife, Jose; Todd Massey, H.; Gertler, Frank B.

2011-01-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena−/−) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena−/− mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena−/− hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena−/− mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction. PMID:21335464

Mammalian enabled (Mena) is a critical regulator of cardiac function.

PubMed

Aguilar, Frédérick; Belmonte, Stephen L; Ram, Rashmi; Noujaim, Sami F; Dunaevsky, Olga; Protack, Tricia L; Jalife, Jose; Todd Massey, H; Gertler, Frank B; Blaxall, Burns C

2011-05-01

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

Role of Specificity Protein-1 and Activating Protein-2 Transcription Factors in the Regulation of the Gap Junction Protein Beta-2 Gene in the Epididymis of the Rat.

PubMed

Adam, Cécile; Cyr, Daniel G

2016-06-01

In prepubertal rats, connexin 26 (GJB2) is expressed between adjacent columnar cells of the epididymis. At 28 days of age, when columnar cells differentiate into adult epithelial cell types, Gjb2 mRNA levels decrease to barely detectable levels. There is no information on the regulation of GJB2 in the epididymis. The present study characterized regulation of the Gjb2 gene promoter in the epididymis. A single transcription start site at position -3829 bp relative to the ATG was identified. Computational analysis revealed several TFAP2A, SP1, and KLF4 putative binding sites. A 1.5-kb fragment of the Gjb2 promoter was cloned into a vector containing a luciferase reporter gene. Transfection of the construct into immortalized rat caput epididymal (RCE-1) cells indicated that the promoter contained sufficient information to drive expression of the reporter gene. Deletion constructs showed that the basal activity of the promoter resides in the first -230 bp of the transcriptional start site. Two response elements necessary for GJB2 expression were identified: an overlapping TFAP2A/SP1 site (-136 to -126 bp) and an SP1 site (-50 bp). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays confirmed that SP1 and TFAP2A were bound to the promoter. ChIP analysis of chromatin from young and pubertal rats indicated that TFAP2A and SP1 binding decreased with age. SP1 and TFAP2A knockdown indicated that SP1 is necessary for Gjb2 expression. DNA methylation did not appear to be involved in the regulation of Gjb2 expression. Results indicate that SP1 and TFAP2A regulate Gjb2 promoter activity during epididymal differentiation in rat. © 2016 by the Society for the Study of Reproduction, Inc.

Floral gene resources from basal angiosperms for comparative genomics research

PubMed Central

Albert, Victor A; Soltis, Douglas E; Carlson, John E; Farmerie, William G; Wall, P Kerr; Ilut, Daniel C; Solow, Teri M; Mueller, Lukas A; Landherr, Lena L; Hu, Yi; Buzgo, Matyas; Kim, Sangtae; Yoo, Mi-Jeong; Frohlich, Michael W; Perl-Treves, Rafael; Schlarbaum, Scott E; Bliss, Barbara J; Zhang, Xiaohong; Tanksley, Steven D; Oppenheimer, David G; Soltis, Pamela S; Ma, Hong; dePamphilis, Claude W; Leebens-Mack, James H

2005-01-01

Background The Floral Genome Project was initiated to bridge the genomic gap between the most broadly studied plant model systems. Arabidopsis and rice, although now completely sequenced and under intensive comparative genomic investigation, are separated by at least 125 million years of evolutionary time, and cannot in isolation provide a comprehensive perspective on structural and functional aspects of flowering plant genome dynamics. Here we discuss new genomic resources available to the scientific community, comprising cDNA libraries and Expressed Sequence Tag (EST) sequences for a suite of phylogenetically basal angiosperms specifically selected to bridge the evolutionary gaps between model plants and provide insights into gene content and genome structure in the earliest flowering plants. Results Random sequencing of cDNAs from representatives of phylogenetically important eudicot, non-grass monocot, and gymnosperm lineages has so far (as of 12/1/04) generated 70,514 ESTs and 48,170 assembled unigenes. Efficient sorting of EST sequences into putative gene families based on whole Arabidopsis/rice proteome comparison has permitted ready identification of cDNA clones for finished sequencing. Preliminarily, (i) proportions of functional categories among sequenced floral genes seem representative of the entire Arabidopsis transcriptome, (ii) many known floral gene homologues have been captured, and (iii) phylogenetic analyses of ESTs are providing new insights into the process of gene family evolution in relation to the origin and diversification of the angiosperms. Conclusion Initial comparisons illustrate the utility of the EST data sets toward discovery of the basic floral transcriptome. These first findings also afford the opportunity to address a number of conspicuous evolutionary genomic questions, including reproductive organ transcriptome overlap between angiosperms and gymnosperms, genome-wide duplication history, lineage-specific gene duplication and functional divergence, and analyses of adaptive molecular evolution. Since not all genes in the floral transcriptome will be associated with flowering, these EST resources will also be of interest to plant scientists working on other functions, such as photosynthesis, signal transduction, and metabolic pathways. PMID:15799777

Expression of gap junction genes connexin 32 and connexin 43 mRNAs and proteins, and their role in hepatocarcinogenesis

PubMed Central

Ma, Xiang-Dong; Ma, Xing; Sui, Yan-Fang; Wang, Wen-Liang

2002-01-01

AIM: To investigate the relationship between hepatocarcinogenesis and the expression of connexin32 (cx32), connexin43 (cx43) mRNAs and proteins in vitro. METHODS: Gap junction genes cx32 and cx43 mRNA in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal liver cell line QZG were detected by in situ hybridization (ISH) with digoxin-labeled cx32, and cx43 cDNA probes. Expression of Cx32 and Cx43 proteins in the cell lines was revealed by indirect immuno-fluorescence and flow cytometry (FCM). RESULTS: Blue positive hybridization signals of cx32 and cx43 mRNAs detected by ISH with cx32 and cx43 cDNA probes respectively were located in cytoplasm of cells of HHCC, SMMC-7721 and QZG. No significant difference of either cx32 mRNA or cx43 mRNA was tested among HHCC, SMMC-7721 and QZG (P = 2.673, HHCC vs QZG; P = 1.375, SMMC-7721 vs QZG). FCM assay showed that the positive rates of Cx32 protein in HHCC, SMMC-7721 and QZG were 0.7%, 1.7% and 99.0%, and the positive rates of Cx43 protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5% and 99.1% respectively. Significant differences of both Cx32 and Cx43 protein expression existed between hepatocellular carcinoma cell lines and normal liver cell line (P = 0.0069, HHCC vs QZG; P = 0.0087, SMMC-7721 vs QZG). Moreover, the fluorescent intensities of Cx32 and Cx43 proteins in HHCC, SMMC-7721 were lower than that in QZG. CONCLUSION: Hepatocellular carcinoma cell lines HHCC and SMMC-7721 exhibited lower positive rates and fluorescent intensities of Cx32, Cx43 proteins compared with that of normal liver cell line QZG. It is suggested that lower expression of both Cx32 and Cx43 proteins in hepatocellular carcinoma cells could play pivotal roles in the hepatocarcinogenesis. Besides, genetic defects of cx32 and cx43 in post-translational processing should be considered. PMID:11833073

Genome-wide transcriptional profiling data from chronic cutaneous lupus erythematosus (CCLE) peripheral blood.

PubMed

Dey-Rao, R; Sinha, A A

2015-03-01

The disease Lupus erythematosus (LE), exhibits a variety of clinical manifestations with potentially wide-ranging multi-organ damage to joints, tendons, kidney, lung, heart, blood vessels, central nervous system and skin [1,2] Systemic changes are likely to trigger organ specific manifestation of the disease. Here, we provide the data examined to address the gap in knowledge regarding causes and mechanisms contributing to the autoimmune attack on skin in chronic cutaneous lupus erythematosus (CCLE). The raw gene expression data files (CEL files) are provided with this article [3].

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis

PubMed Central

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A.

2012-01-01

Purpose To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. Methods In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9–14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca2+-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Results Significantly lower mean activities of Ca2+-ATPase and Na+/K+ -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca2+-ATPase and Na+/K+-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. Conclusions The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication. PMID:22876134

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PubMed

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A; Geraldine, Pitchairaj

2012-01-01

To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Expression of beta2-microglobulin and c-fos mRNA: is there an influence of high- or low-flux dialyzer membranes?

PubMed

Haufe, C C; Eismann, U; Deppisch, R M; Stein, G

2001-02-01

Dialysis-related amyloidosis is an important complication of long-term hemodialysis (HD) therapy with several pathogenetic factors. One of them is the influence of the dialyzer membrane type on the synthesis of beta2-microglobulin (beta2m). In vitro results are controversial. Thus, the hypothesis of whether in vivo beta2m generation is induced by the HD procedure and whether this induction depends on the type of the used dialyzer membrane should be tested. The aim of the present study was to investigate the influence of "biocompatible" high-flux versus "bioincompatible" low-flux HD on in vivo beta2m generation as well as the induction of the early activation gene c-fos in peripheral blood cells. Six nondiabetic HD patients [mean age 46 (21 to 69) years; Kt/V> 1.2] were included in a randomized crossover study using either a low-flux (cellulosic/cuprophan) or a high-flux (polyamide) dialyzer membrane. At the end of a four-week run-in period for each membrane, whole blood samples were taken before, immediately at, and four hours after the end of the dialysis session. MRNA was extracted, and after transcription to cDNA, quantitative polymerase chain reaction was performed for the beta2m gene, the early response gene c-fos, and the GAP-DH housekeeping gene. Based on the applied method for detection of specific mRNA, the results were given as ratio of beta2m or c-fos cDNA per GAP-DH cDNA. General cell activation during HD was indicated by increasing mRNA expression of c-fos related to the time course of the dialysis session, whereas beta2m did not change significantly. However, no difference was found when comparing the low-flux and the high-flux dialyzer membranes. Despite the evidence for activation of peripheral blood cells, as indicated by increasing c-fos message, no sign of beta2m mRNA induction during HD procedure with different dialyzer membranes was seen. Our results suggest that there is post-transcriptional regulation of beta2m generation and/or release as well as the influence of the dialyzer membrane type on post-translational processes, that is, advance glycation end products (AGE) or conformational modification of the beta2m protein. Furthermore, our data demonstrate that gene expression patterns during dialysis and/or uremia are not homogenous and need to be investigated further, especially with respect to the proinflammatory role of early leukocyte activation signals.

The GTPase Activating Rap/RanGAP Domain-Like 1 Gene Is Associated with Chicken Reproductive Traits

PubMed Central

Shen, Xu; Zeng, Hua; Xie, Liang; He, Jun; Li, Jian; Xie, Xiujuan; Luo, Chenglong; Xu, Haiping; Zhou, Min; Nie, Qinghua; Zhang, Xiquan

2012-01-01

Background Abundant evidence indicates that chicken reproduction is strictly regulated by the hypothalamic-pituitary-gonad (HPG) axis, and the genes included in the HPG axis have been studied extensively. However, the question remains as to whether any other genes outside of the HPG system are involved in regulating chicken reproduction. The present study was aimed to identify, on a genome-wide level, novel genes associated with chicken reproductive traits. Methodology/Principal Finding Suppressive subtractive hybridization (SSH), genome-wide association study (GWAS), and gene-centric GWAS were used to identify novel genes underlying chicken reproduction. Single marker-trait association analysis with a large population and allelic frequency spectrum analysis were used to confirm the effects of candidate genes. Using two full-sib Ningdu Sanhuang (NDH) chickens, GARNL1 was identified as a candidate gene involved in chicken broodiness by SSH analysis. Its expression levels in the hypothalamus and pituitary were significantly higher in brooding chickens than in non-brooding chickens. GWAS analysis with a NDH two tail sample showed that 2802 SNPs were significantly associated with egg number at 300 d of age (EN300). Among the 2802 SNPs, 2 SNPs composed a block overlapping the GARNL1 gene. The gene-centric GWAS analysis with another two tail sample of NDH showed that GARNL1 was strongly associated with EN300 and age at first egg (AFE). Single marker-trait association analysis in 1301 female NDH chickens confirmed that variation in this gene was related to EN300 and AFE. The allelic frequency spectrum of the SNP rs15700989 among 5 different populations supported the above associations. Western blotting, RT-PCR, and qPCR were used to analyze alternative splicing of the GARNL1 gene. RT-PCR detected 5 transcripts and revealed that the transcript, which has a 141 bp insertion, was expressed in a tissue-specific manner. Conclusions/Significance Our findings demonstrate that the GARNL1 gene contributes to chicken reproductive traits. PMID:22496769

Utilizing Social Media to Study Information-Seeking and Ethical Issues in Gene Therapy

PubMed Central

Robillard, Julie M; Whiteley, Louise; Johnson, Thomas Wade; Lim, Jonathan; Wasserman, Wyeth W

2013-01-01

Background The field of gene therapy is rapidly evolving, and while hopes of treating disorders of the central nervous system and ethical concerns have been articulated within the academic community, little is known about views and opinions of different stakeholder groups. Objective To address this gap, we utilized social media to investigate the kind of information public users are seeking about gene therapy and the hopes, concerns, and attitudes they express. Methods We conducted a content analysis of questions containing the keywords “gene therapy” from the Q&A site “Yahoo! Answers” for the 5-year period between 2006 and 2010. From the pool of questions retrieved (N=903), we identified those containing at least one theme related to ethics, environment, economics, law, or society (n=173) and then characterized the content of relevant answers (n=399) through emergent coding. Results The results show that users seek a wide range of information regarding gene therapy, with requests for scientific information and ethical issues at the forefront of enquiry. The question sample reveals high expectations for gene therapy that range from cures for genetic and nongenetic diseases to pre- and postnatal enhancement of physiological attributes. Ethics questions are commonly expressed as fears about the impact of gene therapy on self and society. The answer sample echoes these concerns but further suggests that the acceptability of gene therapy varies depending on the specific application. Conclusions Overall, the findings highlight the powerful role of social media as a rich resource for research into attitudes toward biomedicine and as a platform for knowledge exchange and public engagement for topics relating to health and disease. PMID:23470490

Likelihood-Based Gene Annotations for Gap Filling and Quality Assessment in Genome-Scale Metabolic Models

PubMed Central

Benedict, Matthew N.; Mundy, Michael B.; Henry, Christopher S.; Chia, Nicholas; Price, Nathan D.

2014-01-01

Genome-scale metabolic models provide a powerful means to harness information from genomes to deepen biological insights. With exponentially increasing sequencing capacity, there is an enormous need for automated reconstruction techniques that can provide more accurate models in a short time frame. Current methods for automated metabolic network reconstruction rely on gene and reaction annotations to build draft metabolic networks and algorithms to fill gaps in these networks. However, automated reconstruction is hampered by database inconsistencies, incorrect annotations, and gap filling largely without considering genomic information. Here we develop an approach for applying genomic information to predict alternative functions for genes and estimate their likelihoods from sequence homology. We show that computed likelihood values were significantly higher for annotations found in manually curated metabolic networks than those that were not. We then apply these alternative functional predictions to estimate reaction likelihoods, which are used in a new gap filling approach called likelihood-based gap filling to predict more genomically consistent solutions. To validate the likelihood-based gap filling approach, we applied it to models where essential pathways were removed, finding that likelihood-based gap filling identified more biologically relevant solutions than parsimony-based gap filling approaches. We also demonstrate that models gap filled using likelihood-based gap filling provide greater coverage and genomic consistency with metabolic gene functions compared to parsimony-based approaches. Interestingly, despite these findings, we found that likelihoods did not significantly affect consistency of gap filled models with Biolog and knockout lethality data. This indicates that the phenotype data alone cannot necessarily be used to discriminate between alternative solutions for gap filling and therefore, that the use of other information is necessary to obtain a more accurate network. All described workflows are implemented as part of the DOE Systems Biology Knowledgebase (KBase) and are publicly available via API or command-line web interface. PMID:25329157

BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

PubMed

Lopes, Cátia D F; Gonçalves, Nádia P; Gomes, Carla P; Saraiva, Maria J; Pêgo, Ana P

2017-03-01

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sex specific expression and distribution of small RNAs in papaya.

PubMed

Aryal, Rishi; Jagadeeswaran, Guru; Zheng, Yun; Yu, Qingyi; Sunkar, Ramanjulu; Ming, Ray

2014-01-13

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

PubMed

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J; Wahl, James K; Johnson, Keith R; Mehta, Parmender P

2015-02-20

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

SINEs.

PubMed

Kramerov, Dmitri A; Vassetzky, Nikita S

2011-01-01

Short interspersed elements (SINEs) are mobile genetic elements that invade the genomes of many eukaryotes. Since their discovery about 30 years ago, many gaps in our understanding of the biology and function of SINEs have been filled. This review summarizes the past and recent advances in the studies of SINEs. The structure and origin of SINEs as well as the processes involved in their amplification, transcription, RNA processing, reverse transcription, and integration of a SINE copy into the genome are considered. Then we focus on the significance of SINEs for the host genomes. While these genomic parasites can be deleterious to the cell, the long-term being in the genome has made SINEs a valuable source of genetic variation providing regulatory elements for gene expression, alternative splice sites, polyadenylation signals, and even functional RNA genes. Copyright © 2011 John Wiley & Sons, Ltd.

Conditional Lineage Ablation to Model Human Diseases

NASA Astrophysics Data System (ADS)

Lee, Paul; Morley, Gregory; Huang, Qian; Fischer, Avi; Seiler, Stephanie; Horner, James W.; Factor, Stephen; Vaidya, Dhananjay; Jalife, Jose; Fishman, Glenn I.

1998-09-01

Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis.

Relationship between connexin expression and gap-junction resistivity in human atrial myocardium.

PubMed

Dhillon, Paramdeep S; Chowdhury, Rasheda A; Patel, Pravina M; Jabr, Rita; Momin, Aziz U; Vecht, Joshua; Gray, Rosaire; Shipolini, Alex; Fry, Christopher H; Peters, Nicholas S

2014-04-01

The relative roles of the gap-junctional proteins connexin40 (Cx40) and connexin43 (Cx43) in determining human atrial myocardial resistivity is unknown. In addressing the hypothesis that changing relative expression of Cx40 and Cx43 underlies an increase in human atrial myocardial resistivity with age, this relationship was investigated by direct ex vivo measurement of gap-junctional resistivity and quantitative connexin immunoblotting and immunohistochemistry. Oil-gap impedance measurements were performed to determine resistivity of the intracellular pathway (Ri), which correlated with total Cx40 quantification by Western blotting (rs=0.64, P<0.01, n=20). Specific gap-junctional resistivity (Rj) correlated not only with Western immunoquantification of Cx40 (rs=0.63, P=0.01, n=20), but also more specifically, with the Cx40 fraction localized to the intercalated disks on immunohistochemical quantification (rs=0.66, P=0.02, n=12). Although Cx43 expression showed no correlation with resistivity values, the proportional expression of the 2 connexins, (Cx40/[Cx40+Cx43]) correlated with Ri and Rj (rs=0.58, P<0.01 for Ri and rs=0.51, P=0.02 for Rj). Advancing age was associated with a rise in Ri (rs=0.77, P<0.0001), Rj (rs=0.65, P<0.001, n=23), Cx40 quantity (rs=0.54, P=0.01, n=20), and Cx40 gap-junction protein per unit area of en face disk (rs=0.61, P=0.02, n=12). Cx40 is associated with human right atrial gap-junctional resistivity such that increased total, gap-junctional, and proportional Cx40 expression increases gap-junctional resistivity. Accordingly, advancing age is associated with an increase in Cx40 expression and a corresponding increase in gap-junctional resistivity. These findings are the first to demonstrate this relationship and a mechanistic explanation for changing atrial conduction and age-related arrhythmic tendency.

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

PubMed

Christen, Verena; Rusconi, Manuel; Crettaz, Pierre; Fent, Karl

2017-06-15

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex.

PubMed

Dere, E; Zheng-Fischhöfer, Q; Viggiano, D; Gironi Carnevale, U A; Ruocco, L A; Zlomuzica, A; Schnichels, M; Willecke, K; Huston, J P; Sadile, A G

2008-05-02

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

[Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase].

PubMed

Firsova, Iu E; Doronina, N V; Trotsenko, Iu A

2004-01-01

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.

Automatic Compilation from High-Level Biologically-Oriented Programming Language to Genetic Regulatory Networks

PubMed Central

Beal, Jacob; Lu, Ting; Weiss, Ron

2011-01-01

Background The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. Methodology/Principal Findings To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes () and latency of the optimized engineered gene networks. Conclusions/Significance Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems. PMID:21850228

Automatic compilation from high-level biologically-oriented programming language to genetic regulatory networks.

PubMed

Beal, Jacob; Lu, Ting; Weiss, Ron

2011-01-01

The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes (~ 50%) and latency of the optimized engineered gene networks. Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems.

Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

PubMed

Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

2016-08-01

Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. © 2015 International Federation for Cell Biology.

A single high dose of dexamethasone increases GAP-43 and synaptophysin in the hippocampus of aged rats.

PubMed

Tesic, Vesna; Perovic, Milka; Zaletel, Ivan; Jovanovic, Mirna; Puskas, Nela; Ruzdijic, Sabera; Kanazir, Selma

2017-11-01

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, has been reported to modulate cognitive performance in both animals and humans. In the present study, we demonstrate the effects of a single high dose of dexamethasone on the expression and distribution of synaptic plasticity-related proteins, growth-associated protein-43 (GAP-43) and synaptophysin, in the hippocampus of 6-, 12-, 18- and 24-month-old rats. Acute dexamethasone treatment significantly altered the expression of GAP-43 at the posttranslational level by modulating the levels of phosphorylated GAP-43 and proteolytic GAP-43-3 fragment. The effect was the most pronounced in the hippocampi of the aged animals. The total GAP-43 protein was increased only in 24-month-old dexamethasone-treated animals, and was concomitant with a decrease in calpain-mediated proteolysis. Moreover, by introducing the gray level co-occurrence matrix method, a form of texture analysis, we were able to reveal the subtle differences in the expression pattern of both GAP-43 and synaptophysin in the hippocampal subfields that were not detected by Western blot analysis alone. Therefore, the current study demonstrates, through a novel combined approach, that dexamethasone treatment significantly affects both GAP-43 and synaptophysin protein expression in the hippocampus of aged rats. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

PubMed

Moriya, Yuki; Yamada, Takuji; Okuda, Shujiro; Nakagawa, Zenichi; Kotera, Masaaki; Tokimatsu, Toshiaki; Kanehisa, Minoru; Goto, Susumu

2016-03-28

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

PubMed Central

Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

2015-01-01

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ. PMID:26240388

Interferon-gamma inhibits intestinal restitution by preventing gap junction communication between enterocytes.

PubMed

Leaphart, Cynthia L; Qureshi, Faisal; Cetin, Selma; Li, Jun; Dubowski, Theresa; Baty, Catherine; Batey, Catherine; Beer-Stolz, Donna; Guo, Fengli; Murray, Sandra A; Hackam, David J

2007-06-01

Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions. NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA. Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution. IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.

Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons.

PubMed

Rash, J E; Yasumura, T; Dudek, F E; Nagy, J I

2001-03-15

The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.

Comparative Mitogenomic Analyses of Praying Mantises (Dictyoptera, Mantodea): Origin and Evolution of Unusual Intergenic Gaps

PubMed Central

Zhang, Hong-Li; Ye, Fei

2017-01-01

Praying mantises are a diverse group of predatory insects. Although some Mantodea mitogenomes have been reported, a comprehensive comparative and evolutionary genomic study is lacking for this group. In the present study, four new mitogenomes were sequenced, annotated, and compared to the previously published mitogenomes of other Mantodea species. Most Mantodea mitogenomes share a typical set of mitochondrial genes and a putative control region (CR). Additionally, and most intriguingly, another large non-coding region (LNC) was detected between trnM and ND2 in all six Paramantini mitogenomes examined. The main section in this common region of Paramantini may have initially originated from the corresponding control region for each species, whereas sequence differences between the LNCs and CRs and phylogenetic analyses indicate that LNC and CR are largely independently evolving. Namely, the LNC (the duplicated CR) may have subsequently degenerated during evolution. Furthermore, evidence suggests that special intergenic gaps have been introduced in some species through gene rearrangement and duplication. These gaps are actually the original abutting sequences of migrated or duplicated genes. Some gaps (G5 and G6) are homologous to the 5' and 3' surrounding regions of the duplicated gene in the original gene order, and another specific gap (G7) has tandem repeats. We analysed the phylogenetic relationships of fifteen Mantodea species using 37 concatenated mitochondrial genes and detected several synapomorphies unique to species in some clades. PMID:28367101

The Receptive-Expressive Gap in the Vocabulary of Young Second-Language Learners: Robustness and Possible Mechanisms

ERIC Educational Resources Information Center

Gibson, Todd A.; Oller, D. Kimbrough; Jarmulowicz, Linda; Ethington, Corinna A.

2012-01-01

Adults and children learning a second language show difficulty accessing expressive vocabulary that appears accessible receptively in their first language (L1). We call this discrepancy the receptive-expressive gap. Kindergarten Spanish (L1)-English (L2) sequential bilinguals were given standardized tests of receptive and expressive vocabulary in…

Angelman syndrome: current understanding and research prospects.

PubMed

Dan, Bernard

2009-11-01

Angelman syndrome is a neurogenetic disorder characterized by developmental delay, severe intellectual disability, absent speech, exuberant behavior with happy demeanor, motor impairment, and epilepsy, due to deficient UBE3A gene expression that may be caused by various abnormalities of chromosome 15. Recent findings in animal models demonstrated altered dendritic spine formation as well as both synaptic [including gamma-aminobutyric acid (GABA)(A) and N-methyl-D-aspartate (NMDA) transmission] and nonsynaptic (including gap junction) influences in various brain regions, including hippocampus and cerebellar cortex. Reversal of selected abnormalities in rescue genetically engineered animal models is encouraging, although it should not be misinterpreted as promising "cure" for affected patients. Much research is still required to fully understand the functional links between lack of UBE3A expression and clinical manifestations of Angelman syndrome. Studies of regulation of UBE3A expression, including imprinting-related methylation, may point to possibilities of therapeutic upregulation. Understanding relevant roles of the gene product might lead to targeted intervention. Further documentation of brain network dynamics, with particular emphasis on hippocampus, thalamocortical, and cerebellar networks is needed, including in a developmental perspective. There is also a need for further clinical research for improving management of problems such as epilepsy, behavior, communication, learning, motor impairment, and sleep disturbances.

The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing.

PubMed

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla A; Diekwisch, Tom; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBN(Δ5-6) truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

The Ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing

PubMed Central

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla; Diekwisch, Thomas G.H.; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNΔ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. PMID:26899203

Immunotoxicology of titanium dioxide and hydroxylated fullerenes engineered nanoparticles in fish models

NASA Astrophysics Data System (ADS)

Jovanovic, Boris

2011-12-01

Nanoparticles have the potential to cause adverse effects on the fish health, but the understanding of the underlying mechanisms is limited. Major task of this dissertation was to connect gaps in current knowledge with a comprehensive sequence of molecular, cellular and organismal responses toward environmentally relevant concentrations of engineered nanoparticles (titanium dioxide -- TiO2 and hydroxylated fullerenes), outlining the interaction with the innate immune system of fish. The research was divided into following steps: 1) create cDNA libraries for the species of fathead minnow (Pimephales promelas); 2) evaluate whether, and how can nanoparticles modulate neutrophil function in P. promelas; 3) determine the changes in expression of standard biomarker genes as a result of nanoparticle treatment; 4) expose the P. promelas to nanoparticles and appraise their survival rate in a bacterial challenge study; 5) assess the impact of nanoparticles on neuro-immunological interface during the early embryogenesis of zebrafish (Danio rerio). It was hypothesized that engineered nanoparticles can cause measurable changes in fish transcriptome, immune response, and disease resistance. The results of this dissertation are: 1) application of environmentally relevant concentration of nanoparticles changed function of fish neutrophils; 2) fish exposed to nano-TiO2 had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2, while expression of interleukin 11 and myeloperoxidase was significantly increased and expression of elastase 2 was significantly decreased in fish exposed to hydroxylated fullerenes; 3) exposure to environmental estimated concentration of nano-TiO2 significantly increased fish mortality during Aeromonas hydrophila challenge. Analysis of nano-TiO 2 distribution in fish organism outlined that the nano-TiO2 is concentrating in the fish kidney and spleen; 4) during the early embryogenesis of D. rerio exposure to nanoparticles caused shifts in gene regulation response patterns. Significant effects on gene regulation were observed on genes involved in circadian rhythm, kinase activity, vesicular transport and immune response.

Difficulties Using Standardized Tests to Identify the Receptive Expressive Gap in Bilingual Children's Vocabularies.

PubMed

Gibson, Todd A; Oller, D Kimbrough; Jarmulowicz, Linda

2018-03-01

Receptive standardized vocabulary scores have been found to be much higher than expressive standardized vocabulary scores in children with Spanish as L1, learning L2 (English) in school (Gibson et al., 2012). Here we present evidence suggesting the receptive-expressive gap may be harder to evaluate than previously thought because widely-used standardized tests may not offer comparable normed scores. Furthermore monolingual Spanish-speaking children tested in Mexico and monolingual English-speaking children in the US showed other, yet different statistically significant discrepancies between receptive and expressive scores. Results suggest comparisons across widely used standardized tests in attempts to assess a receptive-expressive gap are precarious.

Genome sequence comparison reveals a candidate gene involved in male-hermaphrodite differentiation in papaya (Carica papaya) trees.

PubMed

Ueno, Hiroki; Urasaki, Naoya; Natsume, Satoshi; Yoshida, Kentaro; Tarora, Kazuhiko; Shudo, Ayano; Terauchi, Ryohei; Matsumura, Hideo

2015-04-01

The sex type of papaya (Carica papaya) is determined by the pair of sex chromosomes (XX, female; XY, male; and XY(h), hermaphrodite), in which there is a non-recombining genomic region in the Y and Y(h) chromosomes. This region is presumed to be involved in determination of males and hermaphrodites; it is designated as the male-specific region in the Y chromosome (MSY) and the hermaphrodite-specific region in the Y(h) chromosome (HSY). Here, we identified the genes determining male and hermaphrodite sex types by comparing MSY and HSY genomic sequences. In the MSY and HSY genomic regions, we identified 14,528 nucleotide substitutions and 965 short indels with a large gap and two highly diverged regions. In the predicted genes expressed in flower buds, we found no nucleotide differences leading to amino acid changes between the MSY and HSY. However, we found an HSY-specific transposon insertion in a gene (SVP like) showing a similarity to the Short Vegetative Phase (SVP) gene. Study of SVP-like transcripts revealed that the MSY allele encoded an intact protein, while the HSY allele encoded a truncated protein. Our findings demonstrated that the SVP-like gene is a candidate gene for male-hermaphrodite determination in papaya.

Gene networks and developmental context: the importance of understanding complex gene expression patterns in evolution.

PubMed

Signor, Sarah A; Arbeitman, Michelle N; Nuzhdin, Sergey V

2016-05-01

Animal development is the product of distinct components and interactions-genes, regulatory networks, and cells-and it exhibits emergent properties that cannot be inferred from the components in isolation. Often the focus is on the genotype-to-phenotype map, overlooking the process of development that turns one into the other. We propose a move toward micro-evolutionary analysis of development, incorporating new tools that enable cell type resolution and single-cell microscopy. Using the sex determination pathway in Drosophila to illustrate potential avenues of research, we highlight some of the questions that these emerging technologies can address. For example, they provide an unprecedented opportunity to study heterogeneity within cell populations, and the potential to add the dimension of time to gene regulatory network analysis. Challenges still remain in developing methods to analyze this data and to increase the throughput. However this line of research has the potential to bridge the gaps between previously more disparate fields, such as population genetics and development, opening up new avenues of research. © 2016 Wiley Periodicals, Inc.

Life's attractors : understanding developmental systems through reverse engineering and in silico evolution.

PubMed

Jaeger, Johannes; Crombach, Anton

2012-01-01

We propose an approach to evolutionary systems biology which is based on reverse engineering of gene regulatory networks and in silico evolutionary simulations. We infer regulatory parameters for gene networks by fitting computational models to quantitative expression data. This allows us to characterize the regulatory structure and dynamical repertoire of evolving gene regulatory networks with a reasonable amount of experimental and computational effort. We use the resulting network models to identify those regulatory interactions that are conserved, and those that have diverged between different species. Moreover, we use the models obtained by data fitting as starting points for simulations of evolutionary transitions between species. These simulations enable us to investigate whether such transitions are random, or whether they show stereotypical series of regulatory changes which depend on the structure and dynamical repertoire of an evolving network. Finally, we present a case study-the gap gene network in dipterans (flies, midges, and mosquitoes)-to illustrate the practical application of the proposed methodology, and to highlight the kind of biological insights that can be gained by this approach.

Biodosimetry as a New Paradigm for Determination of Radiation Risks and Risk-Mitigation in Astronauts Exposed to Space Radiation

NASA Technical Reports Server (NTRS)

Richmond, Robert; Cruz, Angela; Bors, Karen

2004-01-01

Predicting risk of cancer in astronauts exposed to space radiation is challenging partly because uncertainties of absorption of dose and the processing of dose-related damage at the cellular level degrade the confidence of predicting the expression of cancer. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the macromolecular profiles related to malignant transformation in cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported, This is the demonstration of two dose-responsive field-effects of enhanced protein-expression. In one case, expression of keratin 18 (K18) in cultures of human mammary epithelial cells (HMEC) irradiated with cesium-137 gamma-rays is enhanced following exposure of log phase cells to relatively low doses of 30 to 90 cGy. K18 has been reported by a marker for tumor staging and for apoptosis. In the second case, expression of connexin 43 (Cx43) is increased in irradiated stationary phase cultures of HMEC, indicating enhanced formation of gap junctions. Gap junctions have been reported to be involved in bystander effects following irradiation. It is a biodosimeter for assessing radiogenic damage. It is suggested further that such biomolecular dosimetry may introduce a new paradigm for assessing cancer risk and risk-mitigation in individuals, a requirement for managing radiation health in astronauts during extended missions in space. This new paradigm is built upon the statistical power provided by the use of functional genomics and proteomics represented in combined gene- and protein-expression assays.

Early Programming of Uterine Tissue by Bisphenol A: Critical Evaluation of Evidence from Animal Exposure Studies

PubMed Central

Suvorov, Alexander; Waxman, David J.

2015-01-01

Exposure to Bisphenol A (BPA) during the critical window of uterine development has been proposed to program the uterus for increased disease susceptibility based on well-documented effects of the potent xenoestrogen diethylstilbestrol. To investigate this proposal, we reviewed 37 studies of prenatal and/or perinatal BPA exposure in animal models and evaluated evidence for: molecular signatures of early BPA exposure; the development of adverse uterine health effects; and epigenetic changes linked to long-term dysregulation of uterine gene expression and health effects. We found substantial evidence for adult uterine effects of early BPA exposure. In contrast, experimental support for epigenetic actions of early BPA exposure is very limited, and largely consists of effects on Hoxa gene DNA methylation. Critical knowledge gaps were identified, including the need to fully characterize short-term and long-term uterine gene responses, interactions with estrogens and other endogenous hormones, and any long-lasting epigenetic signatures that impact adult disease. PMID:26028543

Prospects and challenges for fungal metatranscriptomes of complex communities

DOE PAGES

Kuske, Cheryl Rae; Hesse, Cedar Nelson; Challacombe, Jean Faust; ...

2015-01-22

We report that the ability to extract and purify messenger RNA directly from plants, decomposing organic matter and soil, followed by high-throughput sequencing of the pool of expressed genes, has spawned the emerging research area of metatranscriptomics. Each metatranscriptome provides a snapshot of the composition and relative abundance of actively transcribed genes, and thus provides an assessment of the interactions between soil microorganisms and plants, and collective microbial metabolic processes in many environments. We highlight current approaches for analysis of fungal transcriptome and metatranscriptome datasets across a gradient of community complexity, and note benefits and pitfalls associated with those approaches.more » Finally, we discuss knowledge gaps that limit our current ability to interpret metatranscriptome datasets and suggest future research directions that will require concerted efforts within the scientific community.« less

Transcriptomic characterization of MRI contrast with focus on the T1-w/T2-w ratio in the cerebral cortex.

PubMed

Ritchie, Jacob; Pantazatos, Spiro P; French, Leon

2018-07-01

Magnetic resonance (MR) images of the brain are of immense clinical and research utility. At the atomic and subatomic levels, the sources of MR signals are well understood. However, we lack a comprehensive understanding of the macromolecular correlates of MR signal contrast. To address this gap, we used genome-wide measurements to correlate gene expression with MR signal intensity across the cerebral cortex in the Allen Human Brain Atlas (AHBA). We focused on the ratio of T1-weighted and T2-weighted intensities (T1-w/T2-w ratio image), which is considered to be a useful proxy for myelin content. As expected, we found enrichment of positive correlations between myelin-associated genes and the ratio image, supporting its use as a myelin marker. Genome-wide, there was an association with protein mass, with genes coding for heavier proteins expressed in regions with high T1-w/T2-w values. Oligodendrocyte gene markers were strongly correlated with the T1-w/T2-w ratio, but this was not driven by myelin-associated genes. Mitochondrial genes exhibit the strongest relationship, showing higher expression in regions with low T1-w/T2-w ratio. This may be due to the pH gradient in mitochondria as genes up-regulated by pH in the brain were also highly correlated with the ratio. While we corroborate associations with myelin and synaptic plasticity, differences in the T1-w/T2-w ratio across the cortex are more strongly linked to molecule size, oligodendrocyte markers, mitochondria, and pH. We evaluate correlations between AHBA transcriptomic measurements and a group averaged T1-w/T2-w ratio image, showing agreement with in-sample results. Expanding our analysis to the whole brain results in strong positive T1-w/T2-w correlations for immune system, inflammatory disease, and microglia marker genes. Genes with negative correlations were enriched for neuron markers and synaptic plasticity genes. Lastly, our findings are similar when performed on T1-w or inverted T2-w intensities alone. These results provide a molecular characterization of MR contrast that will aid interpretation of future MR studies of the brain. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Transcriptome Analysis of Green Peach Aphid (Myzus persicae): Insight into Developmental Regulation and Inter-Species Divergence

PubMed Central

Ji, Rui; Wang, Yujun; Cheng, Yanbin; Zhang, Meiping; Zhang, Hong-Bin; Zhu, Li; Fang, Jichao; Zhu-Salzman, Keyan

2016-01-01

Green peach aphid (Myzus persicae) and pea aphid (Acyrthosiphon pisum) are two phylogenetically closely related agricultural pests. While pea aphid is restricted to Fabaceae, green peach aphid feeds on hundreds of plant species from more than 40 families. Transcriptome comparison could shed light on the genetic factors underlying the difference in host range between the two species. Furthermore, a large scale study contrasting gene expression between immature nymphs and fully developed adult aphids would fill a previous knowledge gap. Here, we obtained transcriptomic sequences of green peach aphid nymphs and adults, respectively, using Illumina sequencing technology. A total of 2244 genes were found to be differentially expressed between the two developmental stages, many of which were associated with detoxification, hormone production, cuticle formation, metabolism, food digestion, and absorption. When searched against publically available pea aphid mRNA sequences, 13,752 unigenes were found to have no homologous counterparts. Interestingly, many of these unigenes that could be annotated in other databases were involved in the “xenobiotics biodegradation and metabolism” pathway, suggesting the two aphids differ in their adaptation to secondary metabolites of host plants. Conversely, 3989 orthologous gene pairs between the two species were subjected to calculations of synonymous and nonsynonymous substitutions, and 148 of the genes potentially evolved in response to positive selection. Some of these genes were predicted to be associated with insect-plant interactions. Our study has revealed certain molecular events related to aphid development, and provided some insight into biological variations in two aphid species, possibly as a result of host plant adaptation. PMID:27812361

A Genomic View of the Sea Urchin Nervous System

PubMed Central

Burke, RD; Angerer, LM; Elphick, MR; Humphrey, GW; Yaguchi, S; Kiyama, T; Liang, S; Mu, X; Agca, C; Klein, WH; Brandhorst, BP; Rowe, M; Wilson, K; Churcher, AM; Taylor, JS; Chen, N; Murray, G; Wang, D; Mellott, D; Olinski, R; Hallböök, F; Thorndyke, MC

2007-01-01

The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones, and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins. PMID:16965768

Structural and transcriptional characterization of a novel member of the soybean urease gene family.

PubMed

Wiebke-Strohm, Beatriz; Ligabue-Braun, Rodrigo; Rechenmacher, Ciliana; De Oliveira-Busatto, Luisa Abruzzi; Carlini, Célia Regina; Bodanese-Zanettini, Maria Helena

2016-04-01

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Gene Editing Vectors for Studying Nicotinic Acetylcholine Receptors in Cholinergic Transmission.

PubMed

Peng, Can; Yan, Yijin; Kim, Veronica J; Engle, Staci E; Berry, Jennifer N; McIntosh, J Michael; Neve, Rachael L; Drenan, Ryan M

2018-05-19

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. Production of loss-of-function insertions or deletions (indels) by these "all-in-one" HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT) positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug.

PubMed

Nieto, Alma; Pérez Ishiwara, David G; Orozco, Esther; Sánchez Monroy, Virginia; Gómez García, Consuelo

2017-01-01

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens.

PubMed

Bitel, Claudine L; Perrone-Bizzozero, Nora I; Frederikse, Peter H

2010-11-04

An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as 'global regulators' of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses>2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT-PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype.

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens

PubMed Central

Bitel, Claudine L.; Perrone-Bizzozero, Nora I.

2010-01-01

Purpose An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as ‘global regulators’ of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses >2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. Methods Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT–PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). Results We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. Conclusions The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype. PMID:21139978

Diapause in a tropical oil-collecting bee: molecular basis unveiled by RNA-Seq.

PubMed

Santos, Priscila Karla F; de Souza Araujo, Natalia; Françoso, Elaine; Zuntini, Alexandre Rizzo; Arias, Maria Cristina

2018-04-27

Diapause is a natural phenomenon characterized by an arrest in development that ensures the survival of organisms under extreme environmental conditions. The process has been well documented in arthropods. However, its molecular basis has been mainly studied in species from temperate zones, leaving a knowledge gap of this phenomenon in tropical species. In the present study, the Neotropical and solitary bee Tetrapedia diversipes was employed as a model for investigating diapause in species from tropical zones. Being a bivoltine insect, Tetrapedia diversipes produce two generations of offspring per year. The first generation, normally born during the wet season, develops faster than individuals from the second generation, born after the dry season. Furthermore, it has been shown that the development of the progeny, of the second generation, is halted at the 5th larval instar, and remains in larval diapause during the dry season. Towards the goal of gaining a better understanding of the diapause phenomenon we compared the global gene expression pattern, in larvae, from both reproductive generations and during diapause. The results demonstrate that there are similarities in the observed gene expression patterns to those already described for temperate climate models, and also identify diapause-related genes that have not been previously reported in the literature. The RNA-Seq analysis identified 2275 differentially expressed transcripts, of which 1167 were annotated. Of these genes, during diapause, 352 were upregulated and 815 were downregulated. According to their biological functions, these genes were categorized into the following groups: cellular detoxification, cytoskeleton, cuticle, sterol and lipid metabolism, cell cycle, heat shock proteins, immune response, circadian clock, and epigenetic control. Many of the identified genes have already been described as being related to diapause; however, new genes were discovered, for the first time, in this study. Among those, we highlight: Niemann-Pick type C1, NPC2 and Acyl-CoA binding protein homolog (all involved in ecdysteroid synthesis); RhoBTB2 and SASH1 (associated with cell cycle regulation) and Histone acetyltransferase KAT7 (related to epigenetic transcriptional regulation). The results presented here add important findings to the understanding of diapause in tropical species, thus increasing the comprehension of diapause-related molecular mechanisms.

GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2.

PubMed

Zhao, Chunmei; Ma, Hong; Bossy-Wetzel, Ella; Lipton, Stuart A; Zhang, Zhuohua; Feng, Gen-Sheng

2003-09-05

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.

LGR5/GPR49 is implicated in motor neuron specification in nervous system.

PubMed

Song, Shao-jun; Mao, Xing-gang; Wang, Chao; Han, An-guo; Yan, Ming; Xue, Xiao-yan

2015-01-01

The biological roles of stem cell marker LGR5, the receptor for the Wnt-agonistic R-spondins, for nervous system are poorly known. Bioinformatics analysis in normal human brain tissues revealed that LGR5 is closely related with neuron development and functions. Interestingly, LGR5 and its ligands R-spondins (RSPO2 and RSPO3) are specifically highly expressed in projection motor neurons in the spinal cord, brain stem and cerebral. Inhibition of Notch activity in neural stem cells (NSCs) increased the percentage of neuronal cells and promoted LGR5 expression, while activation of Notch signal decreased neuronal cells and inhibited the LGR5 expression. Furthermore, knockdown of LGR5 inhibited the expression of neuronal markers MAP2, NeuN, GAP43, SYP and CHRM3, and also reduced the expression of genes that program the identity of motor neurons, including Isl1, Lhx3, PHOX2A, TBX20 and NEUROG2. Our data demonstrated that LGR5 is highly expressed in motor neurons in nervous system and is involved in their development by regulating transcription factors that program motor neuron identity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

p120Ras-GAP binds the DLC1 Rho-GAP tumor suppressor protein and inhibits its RhoA GTPase and growth-suppressing activities.

PubMed

Yang, X-Y; Guan, M; Vigil, D; Der, C J; Lowy, D R; Popescu, N C

2009-03-19

DLC1 (deleted in liver cancer 1), which encodes a Rho GTPase-activating protein (Rho-GAP), is a potent tumor suppressor gene that is frequently inactivated in several human cancers. DLC1 is a multidomain protein that has been shown previously to bind members of the tensin gene family. Here we show that p120Ras-GAP (Ras-GAP; also known as RASA1) interacts and extensively colocalizes with DLC1 in focal adhesions. The binding was mapped to the SH3 domain located in the N terminus of Ras-GAP and to the Rho-GAP catalytic domain located in the C terminus of the DLC1. In vitro analyses with purified proteins determined that the isolated Ras-GAP SH3 domain inhibits DLC1 Rho-GAP activity, suggesting that Ras-GAP is a negative regulator of DLC1 Rho-GAP activity. Consistent with this possibility, we found that ectopic overexpression of Ras-GAP in a Ras-GAP-insensitive tumor line impaired the growth-suppressing activity of DLC1 and increased RhoA activity in vivo. Our observations expand the complexity of proteins that regulate DLC1 function and define a novel mechanism of the cross talk between Ras and Rho GTPases.1R01CA129610

Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family.

PubMed

Habenicht, A; Quesada, A; Cerff, R

1997-10-01

A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Mecp2 Mediates Experience-Dependent Transcriptional Upregulation of Ryanodine Receptor Type-3.

PubMed

Torres, Rodrigo F; Hidalgo, Cecilia; Kerr, Bredford

2017-01-01

Mecp2 is a DNA methylation reader that plays a critical role in experience-dependent plasticity. Increasing evidence supports a role for epigenetic modifications in activity-induced gene expression. Hence, candidate genes related to such phenomena are of great interest. Ryanodine receptors are intracellular calcium channels that contribute to hippocampal synaptic plasticity, dendritic spine remodeling, and participate in learning and memory processes. Here we exposed mice to the enriched environment (EE) paradigm, which through increased stimulation induces experience dependent-plasticity, to explore a role for methyl-cytosines, and Mecp2 in directing Ryanodine receptor 3 ( Ryr3 ) transcriptional activity. EE induced a hippocampal-specific increase in the methylation of discrete cytosines located at a Ryr3 isoform promoter; chromatin immunoprecipitation experiments revealed that EE increased Mecp2 binding to this Ryr3 isoform promoter. Interestingly, the experimental paradigm induced robust Ryr3 upregulation, accompanied by miR132 -dependent suppression of p250GAP , a pathway driving synaptogenesis. In contrast to WT mice, Mecp2-null mice showed diminished levels of Ryr3 and displayed impaired EE-induced Ryr3 upregulation, compromising miR132 dependent suppression of p250GAP and experience-dependent structural plasticity. Based on these results, we propose that Mecp2 acts as a transcriptional activator of Ryr3 , contributing to experience-dependent plasticity.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

PubMed

Hirayama, Mio; Kobayashi, Daiki; Mizuguchi, Souhei; Morikawa, Takashi; Nagayama, Megumi; Midorikawa, Uichi; Wilson, Masayo M; Nambu, Akiko N; Yoshizawa, Akiyasu C; Kawano, Shin; Araki, Norie

2013-05-01

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

PubMed Central

Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

2009-01-01

Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an important step towards the identification of all genes in the citrus genome. Furthermore, public availability of the cDNA clones generated in this study, and not only their sequence, enables testing of the biological function of the genes represented in the collection. Expression of the citrus SEP3 homologue, CitrSEP, in Arabidopsis results in early flowering, along with other phenotypes resembling the over-expression of the Arabidopsis SEPALLATA genes. Our findings suggest that the members of the SEP gene family play similar roles in these quite distant plant species. PMID:19747386

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

DOE Office of Scientific and Technical Information (OSTI.GOV)

McIlwain, Sean J.; Peris, Davis; Sardi, Maria

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assemblymore » approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.« less

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

DOE PAGES

McIlwain, Sean J.; Peris, Davis; Sardi, Maria; ...

2016-04-20

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assemblymore » approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.« less

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

PubMed

Wang, Hong-Xing; Gillio-Meina, Carolina; Chen, Shuli; Gong, Xiang-Qun; Li, Tony Y; Bai, Donglin; Kidder, Gerald M

2013-08-01

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia.

PubMed

Guo, Xiaochuan; Hamilton, Peter J; Reish, Nicholas J; Sweatt, J David; Miller, Courtney A; Rumbaugh, Gavin

2009-06-01

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

PubMed Central

Fontes, Joseph D.; Ramsey, Jon; Polk, Jeremy M; Koop, Andre; Denisova, Janna V.; Belousov, Andrei B.

2015-01-01

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. PMID:26017008

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 5: intercellular junctions and contacts between germs cells and Sertoli cells and their regulatory interactions, testicular cholesterol, and genes/proteins associated with more than one germ cell generation.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

In the testis, cell adhesion and junctional molecules permit specific interactions and intracellular communication between germ and Sertoli cells and apposed Sertoli cells. Among the many adhesion family of proteins, NCAM, nectin and nectin-like, catenins, and cadherens will be discussed, along with gap junctions between germ and Sertoli cells and the many members of the connexin family. The blood-testis barrier separates the haploid spermatids from blood borne elements. In the barrier, the intercellular junctions consist of many proteins such as occludin, tricellulin, and claudins. Changes in the expression of cell adhesion molecules are also an essential part of the mechanism that allows germ cells to move from the basal compartment of the seminiferous tubule to the adluminal compartment thus crossing the blood-testis barrier and well-defined proteins have been shown to assist in this process. Several structural components show interactions between germ cells to Sertoli cells such as the ectoplasmic specialization which are more closely related to Sertoli cells and tubulobulbar complexes that are processes of elongating spermatids embedded into Sertoli cells. Germ cells also modify several Sertoli functions and this also appears to be the case for residual bodies. Cholesterol plays a significant role during spermatogenesis and is essential for germ cell development. Lastly, we list genes/proteins that are expressed not only in any one specific generation of germ cells but across more than one generation. Copyright 2009 Wiley-Liss, Inc.

Activation of ERK1/2 and PI3K/Akt by IGF-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by glutamate in vitro.

PubMed

Liu, Zhen; Cai, Heng; Zhang, Ping; Li, Hao; Liu, Huaxiang; Li, Zhenzhong

2012-03-01

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 influences growth-associated protein 43 (GAP-43) expression and activates the extracellular signal-regulated protein kinase (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in DRG neurons with excitotoxicity induced by glutamate (Glu) remains unknown. In this study, embryonic 15-day-old rat DRG explants were cultured for 48 h and then exposed to IGF-1, Glu, Glu + IGF-1, Glu + IGF-1 + PD98059, Glu + IGF-1 + LY294002, Glu + IGF-1 + PD98059 + LY294002 for additional 12 h. The DRG explants were continuously exposed to growth media as control. The levels of GAP-43 mRNA were detected by real time-PCR analysis. The protein levels of GAP-43, phosphorylated ERK1/2, phosphorylated Akt, total ERK1/2, and total Akt were detected by Western blot assay. GAP-43 expression in situ was determined by immunofluorescent labeling. Apoptotic cell death was monitored by Hoechst 33342 staining. IGF-1 alone increased GAP-43 and its mRNA levels in the absence of Glu. The decreased GAP-43 and its mRNA levels caused by Glu could be partially reversed by the presence of IGF-1. IGF-1 rescued neuronal cell death caused by Glu. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. To validate the impact of GAP-43 expression by IGF-1, GAP-43 induction was blocked by administration of dexamethasone (DEX). IGF-1 partially rescued the decrease of GAP-43 and its mRNA levels induced by DEX. DEX induced an increase of cell apoptosis. IGF-1 may play an important role in neuroprotective effects on DRG neurons through regulating GAP-43 expression with excitotoxicity induced by Glu and the process was involved in both ERK1/2 and PI3K/Akt signaling pathways.

Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression.

PubMed

Day, Timothy F; Mewani, Rajshree R; Starr, Joshua; Li, Xin; Chakravarty, Debyani; Ressom, Habtom; Zou, Xiaojun; Eidelman, Ofer; Pollard, Harvey B; Srivastava, Meera; Kasid, Usha N

2017-01-01

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

PubMed

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-08-01

Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

Expression of Key Ion Transporters in the Gill and Esophageal-Gastrointestinal Tract of Euryhaline Mozambique Tilapia Oreochromis mossambicus Acclimated to Fresh Water, Seawater and Hypersaline Water

PubMed Central

Li, Zhengjun; Lui, Eei Yin; Wilson, Jonathan M.; Ip, Yuen Kwong; Lin, Qingsong; Lam, Toong Jin; Lam, Siew Hong

2014-01-01

The ability of euryhaline Mozambique tilapia to tolerate extreme environmental salinities makes it an excellent model for investigating iono-regulation. This study aimed to characterize and fill important information gap of the expression levels of key ion transporters for Na+ and Cl− in the gill and esophageal-gastrointestinal tract of Mozambique tilapia acclimated to freshwater (0 ppt), seawater (30 ppt) and hypersaline (70 ppt) environments. Among the seven genes studied, it was found that nkcc2, nkcc1a, cftr, nka-α1 and nka-α3, were more responsive to salinity challenge than nkcc1b and ncc within the investigated tissues. The ncc expression was restricted to gills of freshwater-acclimated fish while nkcc2 expression was restricted to intestinal segments irrespective of salinity challenge. Among the tissues investigated, gill and posterior intestine were found to be highly responsive to salinity changes, followed by anterior and middle intestine. Both esophagus and stomach displayed significant up-regulation of nka-α1 and nka-α3, but not nkcc isoforms and cftr, in hypersaline-acclimated fish suggesting a response to hypersalinity challenge and involvement of other forms of transporters in iono-regulation. Changes in gene expression levels were partly corroborated by immunohistochemical localization of transport proteins. Apical expression of Ncc was found in Nka-immunoreactive cells in freshwater-acclimated gills while Nkcc co-localized with Nka-immunoreactive cells expressing Cftr apically in seawater- and hypersaline-acclimated gills. In the intestine, Nkcc-stained apical brush border was found in Nka-immunoreactive cells at greater levels under hypersaline conditions. These findings provided new insights into the responsiveness of these genes and tissues under hypersalinity challenge, specifically the posterior intestine being vital for salt absorption and iono-osmoregulation in the Mozambique tilapia; its ability to survive in hypersalinity may be in part related to its ability to up-regulate key ion transporters in the posterior intestine. The findings pave the way for future iono-regulatory studies on the Mozambique tilapia esophageal-gastrointestinal tract. PMID:24498146

Thrombospondin-1, -2 and -5 have differential effects on vascular smooth muscle cell physiology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helkin, Alex; Maier, Kristopher G.; Department of Veterans Affairs VA Healthcare Network Upstate New York at Syracuse, Syracuse, NY

Introduction: The thrombospondins (TSPs) are matricellular proteins that exert multifunctional effects by binding cytokines, cell-surface receptors and other proteins. TSPs play important roles in vascular pathobiology and are all expressed in arterial lesions. The differential effects of TSP-1, -2, and -5 represent a gap in knowledge in vascular smooth muscle cell (VSMC) physiology. Our objective is to determine if structural differences of the TSPs imparted different effects on VSMC functions critical to the formation of neointimal hyperplasia. We hypothesize that TSP-1 and -2 induce similar patterns of migration, proliferation and gene expression, while the effects of TSP-5 are different. Methods:more » Human aortic VSMC chemotaxis was tested for TSP-2 and TSP-5 (1–40 μg/mL), and compared to TSP-1 and serum-free media (SFM) using a modified Boyden chamber. Next, VSMCs were exposed to TSP-1, TSP-2 or TSP-5 (0.2–40 μg/mL). Proliferation was assessed by MTS assay. Finally, VSMCs were exposed to TSP-1, TSP-2, TSP-5 or SFM for 3, 6 or 24 h. Quantitative real-time PCR was performed on 96 genes using a microfluidic card. Statistical analysis was performed by ANOVA or t-test, with p < 0.05 being significant. Results: TSP-1, TSP-2 and TSP-5 at 20 μg/mL all induce chemotaxis 3.1 fold compared to serum-free media. TSP-1 and TSP-2 induced proliferation 53% and 54% respectively, whereas TSP-5 did not. In the gene analysis, overall, cardiovascular system development and function is the canonical pathway most influenced by TSP treatment, and includes multiple growth factors, cytokines and proteases implicated in cellular migration, proliferation, vasculogenesis, apoptosis and inflammation pathways. Conclusions and relevance: The results of this study indicate TSP-1, -2, and -5 play active roles in VSMC physiology and gene expression. Similarly to TSP-1, VSMC chemotaxis to TSP-2 and -5 is dose-dependent. TSP-1 and -2 induces VSMC proliferation, but TSP-5 does not, likely due conservation of N-terminal domains in TSP-1 and -2. In addition, TSP-1, -2 and -5 significantly affect VSMC gene expression; however, little overlap exists in the specific genes altered. This study further delineates TSP-1, -2 and -5's contributions to processes related to VSMC physiology. - Highlights: • We examined the effects of three different thrombospondins on smooth muscle cells. • Thrombospondins −1, −2, −5 all increase smooth muscle cell migration. • Thrombospondins −1 and −2, but not −5, increase smooth muscle cell proliferation. • All three thrombospondins exhibit temporally distinct patterns of gene expression. • Thrombospondins −1 and −2 display distinct patterns of gene expression.« less

FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival

PubMed Central

Heim, Lisanne; Trump, Sonja; Mittler, Susanne; Sopel, Nina; Andreev, Katerina; Ferrazzi, Fulvia; Ekici, Arif B.; Rieker, Ralf; Springel, Rebekka; Assmann, Vera L.; Lechmann, Matthias; Koch, Sonja; Engelhardt, Marina; Trufa, Denis I.; Sirbu, Horia; Hartmann, Arndt; Finotto, Susetta

2017-01-01

ABSTRACT Genome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFβ, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFβ levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFβ accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFβ and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression. PMID:28197372

Role of connexins in metastatic breast cancer and melanoma brain colonization

PubMed Central

Stoletov, Konstantin; Strnadel, Jan; Zardouzian, Erin; Momiyama, Masashi; Park, Frederick D.; Kelber, Jonathan A.; Pizzo, Donald P.; Hoffman, Robert; VandenBerg, Scott R.; Klemke, Richard L.

2013-01-01

Summary Breast cancer and melanoma cells commonly metastasize to the brain using homing mechanisms that are poorly understood. Cancer patients with brain metastases display poor prognosis and survival due to the lack of effective therapeutics and treatment strategies. Recent work using intravital microscopy and preclinical animal models indicates that metastatic cells colonize the brain, specifically in close contact with the existing brain vasculature. However, it is not known how contact with the vascular niche promotes microtumor formation. Here, we investigate the role of connexins in mediating early events in brain colonization using transparent zebrafish and chicken embryo models of brain metastasis. We provide evidence that breast cancer and melanoma cells utilize connexin gap junction proteins (Cx43, Cx26) to initiate brain metastatic lesion formation in association with the vasculature. RNAi depletion of connexins or pharmacological blocking of connexin-mediated cell–cell communication with carbenoxolone inhibited brain colonization by blocking tumor cell extravasation and blood vessel co-option. Activation of the metastatic gene twist in breast cancer cells increased Cx43 protein expression and gap junction communication, leading to increased extravasation, blood vessel co-option and brain colonization. Conversely, inhibiting twist activity reduced Cx43-mediated gap junction coupling and brain colonization. Database analyses of patient histories revealed increased expression of Cx26 and Cx43 in primary melanoma and breast cancer tumors, respectively, which correlated with increased cancer recurrence and metastasis. Together, our data indicate that Cx43 and Cx26 mediate cancer cell metastasis to the brain and suggest that connexins might be exploited therapeutically to benefit cancer patients with metastatic disease. PMID:23321642

Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

PubMed

Vliagoftis, Harissios; Ebeling, Cory; Ilarraza, Ramses; Mahmudi-Azer, Salahaddin; Abel, Melanie; Adamko, Darryl; Befus, A Dean; Moqbel, Redwan

2014-01-01

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

NASA Technical Reports Server (NTRS)

Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

2008-01-01

Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

BREAST CANCER-INDUCED BONE REMODELING, SKELETAL PAIN AND SPROUTING OF SENSORY NERVE FIBERS

PubMed Central

Bloom, Aaron P.; Jimenez-Andrade, Juan M.; Taylor, Reid N.; Castañeda-Corral, Gabriela; Kaczmarska, Magdalena J.; Freeman, Katie T.; Coughlin, Kathleen A.; Ghilardi, Joseph R.; Kuskowski, Michael A.; Mantyh, Patrick W.

2011-01-01

Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other non-malignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP+) sensory nerve fibers. Nearly all CGRP+ nerve fibers that undergo sprouting also co-express tropomyosin receptor kinase A (TrkA+) and growth associated protein-43 (GAP43+). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP+ / TrkA+ / GAP43+ sensory nerve fibers. PMID:21497141

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

PubMed

Mollereau, C; Simons, M J; Soularue, P; Liners, F; Vassart, G; Meunier, J C; Parmentier, M

1996-08-06

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation

PubMed Central

Richter, Günther H. S.; Plehm, Stephanie; Fasan, Annette; Rössler, Sabine; Unland, Rebekka; Bennani-Baiti, Idriss M.; Hotfilder, Marc; Löwel, Diana; von Luettichau, Irene; Mossbrugger, Ilona; Quintanilla-Martinez, Leticia; Kovar, Heinrich; Staege, Martin S.; Müller-Tidow, Carsten; Burdach, Stefan

2009-01-01

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2−/−γC−/− mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor. PMID:19289832

Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth.

PubMed

Parks, Jason C; Patton, Alyssa L; McCallie, Blair R; Griffin, Darren K; Schoolcraft, William B; Katz-Jaffe, Mandy G

2016-05-01

Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P < 0.05; fold change ≥ 2). Enriched pathway analysis showed Wnt signalling, mitogen-activated protein kinases signalling, focal adhesion and tricarboxylic acid cycle to be affected by implantation outcome. The Wnt/beta-catenin signalling pathway, including genes APC, AXIN and GSK3B, were independently validated by real-time quantitative reverse transcription. Individual, corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Likelihood-based gene annotations for gap filling and quality assessment in genome-scale metabolic models

DOE PAGES

Benedict, Matthew N.; Mundy, Michael B.; Henry, Christopher S.; ...

2014-10-16

Genome-scale metabolic models provide a powerful means to harness information from genomes to deepen biological insights. With exponentially increasing sequencing capacity, there is an enormous need for automated reconstruction techniques that can provide more accurate models in a short time frame. Current methods for automated metabolic network reconstruction rely on gene and reaction annotations to build draft metabolic networks and algorithms to fill gaps in these networks. However, automated reconstruction is hampered by database inconsistencies, incorrect annotations, and gap filling largely without considering genomic information. Here we develop an approach for applying genomic information to predict alternative functions for genesmore » and estimate their likelihoods from sequence homology. We show that computed likelihood values were significantly higher for annotations found in manually curated metabolic networks than those that were not. We then apply these alternative functional predictions to estimate reaction likelihoods, which are used in a new gap filling approach called likelihood-based gap filling to predict more genomically consistent solutions. To validate the likelihood-based gap filling approach, we applied it to models where essential pathways were removed, finding that likelihood-based gap filling identified more biologically relevant solutions than parsimony-based gap filling approaches. We also demonstrate that models gap filled using likelihood-based gap filling provide greater coverage and genomic consistency with metabolic gene functions compared to parsimony-based approaches. Interestingly, despite these findings, we found that likelihoods did not significantly affect consistency of gap filled models with Biolog and knockout lethality data. This indicates that the phenotype data alone cannot necessarily be used to discriminate between alternative solutions for gap filling and therefore, that the use of other information is necessary to obtain a more accurate network. All described workflows are implemented as part of the DOE Systems Biology Knowledgebase (KBase) and are publicly available via API or command-line web interface.« less

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery

PubMed Central

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-01-01

Background DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. Results GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. Conclusion GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at . PMID:19728865

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery.

PubMed

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-09-03

DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at http://cgs.pharm.kyoto-u.ac.jp/services/network.

Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors

PubMed Central

2013-01-01

Background In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Results Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast precursors. Together, these data support a very early gastrula stage segregation of the myogenic lineage. Conclusions From this analysis, we are able to precisely define the regulatory and differentiation signatures of the circumesophageal muscle in the sea urchin embryo. Our findings have important implications in understanding the evolution of development of the muscle cell lineage at the molecular level. The data presented here suggest a high level of conservation of the myogenic specification mechanisms across wide phylogenetic distances, but also reveal clear cases of gene cooption. PMID:24295205

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

PubMed Central

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

Connexin 32 is involved in mitosis.

PubMed

Mones, Saleh; Bordignon, Benoit; Fontes, Michel

2012-03-01

The X-linked form of Charcot-Marie-Tooth disorder (CMTX) is the second most frequent type (15% of CMT forms). It involves the GJB1 gene coding for connexin 32, a protein involved in gap junction formation and function. There is no curative treatment for CMTX. We present data on transgenic lines that was accomplished by inserting a human BAC carrying the GJB1 gene, in which two different mutations in connexin 32 (Cx32) observed in patients were introduced. Investigation of these models implicated Cx32 in the control of mitotic stability. The model in which Gjb1 has been invalidated had the same phenotype. This new function for Cx32 was recently confirmed by results from the Mitocheck program. Locomotor impediment was seen in the behavior of these animals, the severity of which correlated with transgene copy number and RNA expression. Copyright © 2011 Wiley Periodicals, Inc.

Cutoff Finder: A Comprehensive and Straightforward Web Application Enabling Rapid Biomarker Cutoff Optimization

PubMed Central

Budczies, Jan; Klauschen, Frederick; Sinn, Bruno V.; Győrffy, Balázs; Schmitt, Wolfgang D.; Darb-Esfahani, Silvia; Denkert, Carsten

2012-01-01

Gene or protein expression data are usually represented by metric or at least ordinal variables. In order to translate a continuous variable into a clinical decision, it is necessary to determine a cutoff point and to stratify patients into two groups each requiring a different kind of treatment. Currently, there is no standard method or standard software for biomarker cutoff determination. Therefore, we developed Cutoff Finder, a bundle of optimization and visualization methods for cutoff determination that is accessible online. While one of the methods for cutoff optimization is based solely on the distribution of the marker under investigation, other methods optimize the correlation of the dichotomization with respect to an outcome or survival variable. We illustrate the functionality of Cutoff Finder by the analysis of the gene expression of estrogen receptor (ER) and progesterone receptor (PgR) in breast cancer tissues. This distribution of these important markers is analyzed and correlated with immunohistologically determined ER status and distant metastasis free survival. Cutoff Finder is expected to fill a relevant gap in the available biometric software repertoire and will enable faster optimization of new diagnostic biomarkers. The tool can be accessed at http://molpath.charite.de/cutoff. PMID:23251644

Monitoring Transcriptomic Changes in Soil-Grown Roots and Shoots of Arabidopsis thaliana Subjected to a Progressive Drought Stress.

PubMed

Bashir, Khurram; Rasheed, Sultana; Matsui, Akihiro; Iida, Kei; Tanaka, Maho; Seki, Motoaki

2018-01-01

Numerous experiments have been performed in Arabidopsis to monitor changes in gene expression that occur in response to a variety of abiotic and biotic stresses, different growth conditions, and at various developmental stages. In addition, gene expression patterns have also been characterized among wild-type and mutant genotypes. Despite these numerous reports, transcriptional changes occurring in roots of soil-grown plants subjected to a progressive drought stress have remained undocumented. To fill this gap, we established a system that allows one to establish water-deficit conditions and to collect root and shoot samples with minimal damage to the root system. Arabidopsis plants are grown in a ceramic-based granular soil and subjected to progressive drought stress by withholding water. Root and shoot samples were collected separately, RNA was purified, and a microarray analysis of drought-stressed roots and shoots was performed at 0, 1, 3, 5, 7, and 9 days after the onset of drought stress treatment. Here, we describe the detailed protocol used to analyze the transcriptomic changes occurring in roots and shoots of soil-grown Arabidopsis subjected to a progressive drought stress.

Evidence that ganglion cells react to retinal detachment.

PubMed

Coblentz, Francie E; Radeke, Monte J; Lewis, Geoffrey P; Fisher, Steven K

2003-03-01

Growth associated protein 43 (GAP 43) is involved in synapse formation and it is expressed in the retina in a very specific pattern. Although GAP 43 is downregulated at the time of synapse formation, it can be re-expressed following injury such as axotomy or ischemia. Because of this we sought to characterize the expression of GAP 43 after retinal detachment (RD). Immunoblot, immunocytochemical and quantitative polymerase chain reaction (QPCR) techniques were used to assess the level of GAP 43 expression after experimental RD. GAP 43 was localized to three sublaminae of the inner plexiform layer of the normal retina. GAP 43 became upregulated in a subset of retinal ganglion cells following at least 7 days of RD. By immunoblot GAP 43 could be detected by 3 days. QPCR shows the upregulation of GAP 43 message by 6hr of detachment. To further characterize changes in ganglion cells, we used an antibody to neurofilament 70 and 200kDa (NF) proteins. Anti-NF labels horizontal cells, ganglion cell dendrites in the inner plexiform layer, and ganglion cell axons (fasicles) in the normal retina. Following detachment it is upregulated in horizontal cells and ganglion cells. When detached retina was double labelled with anti-GAP 43 and anti-NF, some cells were labelled with both markers, while others labelled with only one. We have previously shown that second order neurons respond to detachment; here we show that third order neurons are responding as well. Cellular remodelling of this type in response to detachment may explain the slow recovery of vision that often occurs after reattachment, or those changes that are often assumed to be permanent.

Neighboring Genes Show Correlated Evolution in Gene Expression

PubMed Central

Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus).

PubMed

Lavrov, Igor; Fox, Lyle; Shen, Jun; Han, Yingchun; Cheng, Jianguo

2016-01-01

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

Bridging the Gaps: the Promise of Omics Studies in Pediatric Exercise Research

PubMed Central

Radom-Aizik, Shlomit; Cooper, Dan M.

2018-01-01

In this review, we highlight promising new discoveries that may generate useful and clinically relevant insights into the mechanisms that link exercise with growth during critical periods of development. Growth in childhood and adolescence is unique among mammals, and is a dynamic process regulated by an evolution of hormonal and inflammatory mediators, age-dependent progression of gene expression, and environmentally modulated epigenetic mechanisms. Many of these same processes likely affect molecular transducers of physical activity. How the molecular signaling associated with growth is synchronized with signaling associated with exercise is poorly understood. Recent advances in “omics,” namely, genomics and epigenetics, metabolomics, and proteomics, now provide exciting approaches and tools that can be used for the first time to address this gap. A biologic definition of “healthy” exercise that links the metabolic transducers of physical activity with parallel processes that regulate growth will transform health policy and guidelines that promote optimal use of physical activity. PMID:27137166

Gap junction blockade induces apoptosis in human endometrial stromal cells.

PubMed

Yu, Jie; Berga, Sarah L; Zou, Wei; Sun, He-Ying; Johnston-MacAnanny, Erika; Yalcinkaya, Tamer; Sidell, Neil; Bagchi, Indrani C; Bagchi, Milan K; Taylor, Robert N

2014-07-01

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies. © 2014 Wiley Periodicals, Inc.

Generating high temperature tolerant transgenic plants: Achievements and challenges.

PubMed

Grover, Anil; Mittal, Dheeraj; Negi, Manisha; Lavania, Dhruv

2013-05-01

Production of plants tolerant to high temperature stress is of immense significance in the light of global warming and climate change. Plant cells respond to high temperature stress by re-programming their genetic machinery for survival and reproduction. High temperature tolerance in transgenic plants has largely been achieved either by over-expressing heat shock protein genes or by altering levels of heat shock factors that regulate expression of heat shock and non-heat shock genes. Apart from heat shock factors, over-expression of other trans-acting factors like DREB2A, bZIP28 and WRKY proteins has proven useful in imparting high temperature tolerance. Besides these, elevating the genetic levels of proteins involved in osmotic adjustment, reactive oxygen species removal, saturation of membrane-associated lipids, photosynthetic reactions, production of polyamines and protein biosynthesis process have yielded positive results in equipping transgenic plants with high temperature tolerance. Cyclic nucleotide gated calcium channel proteins that regulate calcium influxes across the cell membrane have recently been shown to be the key players in induction of high temperature tolerance. The involvement of calmodulins and kinases in activation of heat shock factors has been implicated as an important event in governing high temperature tolerance. Unfilled gaps limiting the production of high temperature tolerant transgenic plants for field level cultivation are discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

PubMed Central

Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

2013-01-01

The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

PubMed Central

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-01-01

BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. PMID:20649601

Neighboring Genes Show Correlated Evolution in Gene Expression.

PubMed

Ghanbarian, Avazeh T; Hurst, Laurence D

2015-07-01

When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Omics of Brucella: Species-Specific sRNA-Mediated Gene Ontology Regulatory Networks Identified by Computational Biology.

PubMed

Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Sridhar, Jayavel; Rajendhran, Jeyaprakash

2016-06-01

Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella.

Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR.

PubMed

Ewen-Campen, Ben; Mohr, Stephanie E; Hu, Yanhui; Perrimon, Norbert

2017-10-09

Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research. Copyright © 2017 Elsevier Inc. All rights reserved.

The Socio-Economic Gradient in Children's Reading Skills and the Role of Genetics

ERIC Educational Resources Information Center

Jerrim, John; Vignoles, Anna; Lingam, Raghu; Friend, Angela

2015-01-01

By the time children leave primary school there is a large socio-economic gap in their reading proficiency. There are a number of potential explanations for this socio-economic gap and in this paper we investigate the role of three particular genes and gene-environment interactions in determining children's reading skills, using the Avon…

Growth and stress response mechanisms underlying post-feeding regenerative organ growth in the Burmese python.

PubMed

Andrew, Audra L; Perry, Blair W; Card, Daren C; Schield, Drew R; Ruggiero, Robert P; McGaugh, Suzanne E; Choudhary, Amit; Secor, Stephen M; Castoe, Todd A

2017-05-02

Previous studies examining post-feeding organ regeneration in the Burmese python (Python molurus bivittatus) have identified thousands of genes that are significantly differentially regulated during this process. However, substantial gaps remain in our understanding of coherent mechanisms and specific growth pathways that underlie these rapid and extensive shifts in organ form and function. Here we addressed these gaps by comparing gene expression in the Burmese python heart, liver, kidney, and small intestine across pre- and post-feeding time points (fasted, one day post-feeding, and four days post-feeding), and by conducting detailed analyses of molecular pathways and predictions of upstream regulatory molecules across these organ systems. Identified enriched canonical pathways and upstream regulators indicate that while downstream transcriptional responses are fairly tissue specific, a suite of core pathways and upstream regulator molecules are shared among responsive tissues. Pathways such as mTOR signaling, PPAR/LXR/RXR signaling, and NRF2-mediated oxidative stress response are significantly differentially regulated in multiple tissues, indicative of cell growth and proliferation along with coordinated cell-protective stress responses. Upstream regulatory molecule analyses identify multiple growth factors, kinase receptors, and transmembrane receptors, both within individual organs and across separate tissues. Downstream transcription factors MYC and SREBF are induced in all tissues. These results suggest that largely divergent patterns of post-feeding gene regulation across tissues are mediated by a core set of higher-level signaling molecules. Consistent enrichment of the NRF2-mediated oxidative stress response indicates this pathway may be particularly important in mediating cellular stress during such extreme regenerative growth.

Genetic variants related to gap junctions and hormone secretion influence conception rates in cows

PubMed Central

Sugimoto, Mayumi; Sasaki, Shinji; Gotoh, Yusaku; Nakamura, Yuuki; Aoyagi, Yoshito; Kawahara, Takayoshi; Sugimoto, Yoshikazu

2013-01-01

The recent decline in fertility is a serious problem in the dairy industry. To overcome this problem, we performed a genome-wide association study using 384 Holsteins and identified four loci associated with conception rates. Two of them contained gap junction-related genes: PKP2 and CTTNBP2NL. Further analysis confirmed that PKP2 increased connexin 43, a gap junction protein, whereas CTTNBP2NL dephosphorylated connexin 43. Knockdown of PKP2 or overexpression of CTTNBP2NL inhibited embryo implantation in mice. The other two loci contained neuroendocrine-related genes: SETD6 and CACNB2. Additional experiments indicated that SETD6 is involved in the transcriptional regulation of gonadotropin-releasing hormone, whereas CACNB2 controlled the secretion of follicle-stimulating hormone in cattle. The total allele substitution effect of these genes on conception rate was 3.5%. Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry. PMID:24218568

Genetic variants related to gap junctions and hormone secretion influence conception rates in cows.

PubMed

Sugimoto, Mayumi; Sasaki, Shinji; Gotoh, Yusaku; Nakamura, Yuuki; Aoyagi, Yoshito; Kawahara, Takayoshi; Sugimoto, Yoshikazu

2013-11-26

The recent decline in fertility is a serious problem in the dairy industry. To overcome this problem, we performed a genome-wide association study using 384 Holsteins and identified four loci associated with conception rates. Two of them contained gap junction-related genes: PKP2 and CTTNBP2NL. Further analysis confirmed that PKP2 increased connexin 43, a gap junction protein, whereas CTTNBP2NL dephosphorylated connexin 43. Knockdown of PKP2 or overexpression of CTTNBP2NL inhibited embryo implantation in mice. The other two loci contained neuroendocrine-related genes: SETD6 and CACNB2. Additional experiments indicated that SETD6 is involved in the transcriptional regulation of gonadotropin-releasing hormone, whereas CACNB2 controlled the secretion of follicle-stimulating hormone in cattle. The total allele substitution effect of these genes on conception rate was 3.5%. Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry.

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christen, Verena

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor andmore » different concentrations of biocides for 5 days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals. - Highlights: • The developmental neurotoxicity (DNT) of biocides is poorly known. • We assessed different classes of biocides for DNT in PC-12 cells. • DNT was shown by neurite outgrowth (NOG) and transcriptional changes. • Azamethiphos, chlorpyrifos, dieldrin and heptachlor showed DNT. • NOG correlated with induction of gap-43 which is an important determinant for DNT.« less

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

PubMed Central

Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A.; Dilly, Suzanne J.; James, Sean; Dimitri, Wade; Ladwa, Sweta R.; Taylor, Paul C.; Singer, Donald R. J.

2016-01-01

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states. PMID:26863535

Application of community phylogenetic approaches to understand gene expression: differential exploration of venom gene space in predatory marine gastropods.

PubMed

Chang, Dan; Duda, Thomas F

2014-06-05

Predatory marine gastropods of the genus Conus exhibit substantial variation in venom composition both within and among species. Apart from mechanisms associated with extensive turnover of gene families and rapid evolution of genes that encode venom components ('conotoxins'), the evolution of distinct conotoxin expression patterns is an additional source of variation that may drive interspecific differences in the utilization of species' 'venom gene space'. To determine the evolution of expression patterns of venom genes of Conus species, we evaluated the expression of A-superfamily conotoxin genes of a set of closely related Conus species by comparing recovered transcripts of A-superfamily genes that were previously identified from the genomes of these species. We modified community phylogenetics approaches to incorporate phylogenetic history and disparity of genes and their expression profiles to determine patterns of venom gene space utilization. Less than half of the A-superfamily gene repertoire of these species is expressed, and only a few orthologous genes are coexpressed among species. Species exhibit substantially distinct expression strategies, with some expressing sets of closely related loci ('under-dispersed' expression of available genes) while others express sets of more disparate genes ('over-dispersed' expression). In addition, expressed genes show higher dN/dS values than either unexpressed or ancestral genes; this implies that expression exposes genes to selection and facilitates rapid evolution of these genes. Few recent lineage-specific gene duplicates are expressed simultaneously, suggesting that expression divergence among redundant gene copies may be established shortly after gene duplication. Our study demonstrates that venom gene space is explored differentially by Conus species, a process that effectively permits the independent and rapid evolution of venoms in these species.

Natural gene expression variation studies in yeast.

PubMed

Thompson, Dawn A; Cubillos, Francisco A

2017-01-01

The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants, mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap, such as mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, probably owing to genetic divergence. Future studies in this area will benefit from the access to a massive array of well-annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Integrated Translatome and Proteome: Approach for Accurate Portraying of Widespread Multifunctional Aspects of Trichoderma

PubMed Central

Sharma, Vivek; Salwan, Richa; Sharma, P. N.; Gulati, Arvind

2017-01-01

Genome-wide studies of transcripts expression help in systematic monitoring of genes and allow targeting of candidate genes for future research. In contrast to relatively stable genomic data, the expression of genes is dynamic and regulated both at time and space level at different level in. The variation in the rate of translation is specific for each protein. Both the inherent nature of an mRNA molecule to be translated and the external environmental stimuli can affect the efficiency of the translation process. In biocontrol agents (BCAs), the molecular response at translational level may represents noise-like response of absolute transcript level and an adaptive response to physiological and pathological situations representing subset of mRNAs population actively translated in a cell. The molecular responses of biocontrol are complex and involve multistage regulation of number of genes. The use of high-throughput techniques has led to rapid increase in volume of transcriptomics data of Trichoderma. In general, almost half of the variations of transcriptome and protein level are due to translational control. Thus, studies are required to integrate raw information from different “omics” approaches for accurate depiction of translational response of BCAs in interaction with plants and plant pathogens. The studies on translational status of only active mRNAs bridging with proteome data will help in accurate characterization of only a subset of mRNAs actively engaged in translation. This review highlights the associated bottlenecks and use of state-of-the-art procedures in addressing the gap to accelerate future accomplishment of biocontrol mechanisms. PMID:28900417

De novo transcriptome sequencing and comparative analysis to discover genes involved in ovarian maturity in Strongylocentrotus nudus.

PubMed

Jia, Zhiying; Wang, Qiai; Wu, Kaikai; Wei, Zhenlin; Zhou, Zunchun; Liu, Xiaolin

2017-09-01

Strongylocentrotus nudus is an edible sea urchin, mainly harvested in China. Correlation studies indicated that S. nudus with larger diameter have a prolonged marketing time and better palatability owing to their precocious gonads and extended maturation process. However, the molecular mechanism underlying this phenomenon is still unknown. Here, transcriptome sequencing was applied to study the ovaries of adult S. nudus with different shell diameters to explore the possible mechanism. In this study, four independent cDNA libraries were constructed, including two from the big size urchins and two from the small ones using a HiSeq™2500 platform. A total of 88,581 unigenes were acquired with a mean length of 1354bp, of which 66,331 (74.88%) unigenes could be annotated using six major publicly available databases. Comparative analysis revealed that 353 unigenes were differentially expressed (with log2(ratio)≥1, FDR≤0.001) between the two groups. Of these, 20 differentially expressed genes (DEGs) were selected to confirm the accuracy of RNA-seq data by quantitative real-time RT-PCR. Furthermore, gene ontology and KEGG pathway enrichment analyses were performed to find the putative genes and pathways related to ovarian maturity. Eight unigenes were identified as significant DEGs involved in reproduction related pathways; these included Mos, Cdc20, Rec8, YP30, cytochrome P450 2U1, ovoperoxidase, proteoliaisin, and rendezvin. Our research fills the gap in the studies on the S. nudus ovaries using transcriptome analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

PubMed

Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

2017-06-01

The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. © 2017 Scandinavian Plant Physiology Society.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug

PubMed Central

Nieto, Alma; Pérez Ishiwara, David G.; Orozco, Esther; Sánchez Monroy, Virginia; Gómez García, Consuelo

2017-01-01

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position −170 to −111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (−151 to −136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug. PMID:29238701

FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data.

PubMed

Manijak, Mieszko P; Nielsen, Henrik B

2011-06-11

Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially circumvented by instead matching gene expression signatures to signatures of other experiments. To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700 Arabidopsis microarray experiments. Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/.

Parsimony and Model-Based Analyses of Indels in Avian Nuclear Genes Reveal Congruent and Incongruent Phylogenetic Signals

PubMed Central

Yuri, Tamaki; Kimball, Rebecca T.; Harshman, John; Bowie, Rauri C. K.; Braun, Michael J.; Chojnowski, Jena L.; Han, Kin-Lan; Hackett, Shannon J.; Huddleston, Christopher J.; Moore, William S.; Reddy, Sushma; Sheldon, Frederick H.; Steadman, David W.; Witt, Christopher C.; Braun, Edward L.

2013-01-01

Insertion/deletion (indel) mutations, which are represented by gaps in multiple sequence alignments, have been used to examine phylogenetic hypotheses for some time. However, most analyses combine gap data with the nucleotide sequences in which they are embedded, probably because most phylogenetic datasets include few gap characters. Here, we report analyses of 12,030 gap characters from an alignment of avian nuclear genes using maximum parsimony (MP) and a simple maximum likelihood (ML) framework. Both trees were similar, and they exhibited almost all of the strongly supported relationships in the nucleotide tree, although neither gap tree supported many relationships that have proven difficult to recover in previous studies. Moreover, independent lines of evidence typically corroborated the nucleotide topology instead of the gap topology when they disagreed, although the number of conflicting nodes with high bootstrap support was limited. Filtering to remove short indels did not substantially reduce homoplasy or reduce conflict. Combined analyses of nucleotides and gaps resulted in the nucleotide topology, but with increased support, suggesting that gap data may prove most useful when analyzed in combination with nucleotide substitutions. PMID:24832669

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

PubMed Central

2014-01-01

Background Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. Results In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3–5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. Conclusions We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions. PMID:24742273

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

PubMed

Várnai, Anikó; Tang, Campbell; Bengtsson, Oskar; Atterton, Andrew; Mathiesen, Geir; Eijsink, Vincent G H

2014-04-18

Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

New insights into roles of cell wall invertase in early seed development revealed by comprehensive spatial and temporal expression patterns of GhCWIN1 in cotton.

PubMed

Wang, Lu; Ruan, Yong-Ling

2012-10-01

Despite substantial evidence on the essential roles of cell wall invertase (CWIN) in seed filling, it remains largely unknown how CWIN exerts its regulation early in seed development, a critical stage that sets yield potential. To fill this knowledge gap, we systematically examined the spatial and temporal expression patterns of a major CWIN gene, GhCWIN1, in cotton (Gossypium hirsutum) seeds from prefertilization to prestorage phase. GhCWIN1 messenger RNA was abundant at the innermost seed coat cell layer at 5 d after anthesis but became undetectable at 10 d after anthesis, at the onset of its differentiation into transfer cells characterized by wall ingrowths, suggesting that CWIN may negatively regulate transfer cell differentiation. Within the filial tissues, GhCWIN1 transcript was detected in endosperm cells undergoing nuclear division but not in those cells at the cellularization stage, with similar results observed in Arabidopsis (Arabidopsis thaliana) endosperm for CWIN, AtCWIN4. These findings indicate a function of CWIN in nuclear division but not cell wall biosynthesis in endosperm, contrasting to the role proposed for sucrose synthase (Sus). Further analyses revealed a preferential expression pattern of GhCWIN1 and AtCWIN4 in the provascular region of the torpedo embryos in cotton and Arabidopsis seed, respectively, indicating a role of CWIN in vascular initiation. Together, these novel findings provide insights into the roles of CWIN in regulating early seed development spatially and temporally. By comparing with previous studies on Sus expression and in conjunction with the expression of other related genes, we propose models of CWIN- and Sus-mediated regulation of early seed development.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss

PubMed Central

de Groot, John C.M.J.; van Iperen, Liesbeth; Huisman, Margriet A.; Frijns, Johan H.M.

2015-01-01

ABSTRACT Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9–W18) and show the cochlear expression dynamics of key potassium‐regulating proteins. At W12, MITF+/SOX10+/KIT+ neural‐crest‐derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+‐ATPase‐positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1219–1240, 2015 PMID:25663387

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells.

PubMed

Nagasawa, Kunihiko; Chiba, Hideki; Fujita, Hiroki; Kojima, Takashi; Saito, Tsuyoshi; Endo, Toshiaki; Sawada, Norimasa

2006-07-01

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed. Copyright 2006 Wiley-Liss, Inc.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

PKC-mediated HuD-GAP43 pathway activation in a mouse model of antiretroviral painful neuropathy.

PubMed

Sanna, M D; Quattrone, A; Ghelardini, C; Galeotti, N

2014-03-01

Patients treated with nucleoside reverse transcriptase inhibitors (NRTIs) develop painful neuropathies that lead to discontinuation of antiretroviral therapy thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathy are not known. In order to elucidate the mechanisms underlying this drug-induced neuropathy, we have characterized cellular events in the central nervous system following antiretroviral treatment. Systemic administration of the antiretroviral agent, 2',3'-dideoxycytidine (ddC) considerably increased the expression and phosphorylation of protein kinase C (PKC) γ and ɛ, enzymes highly involved in pain processes, within periaqueductal grey matter (PAG), and, to a lesser extent, within thalamus and prefrontal cortex. These events appeared in coincidence with thermal and mechanical allodynia, but PKC blockade did not prevent the antiretroviral-induced pain hypersensitivity, ruling out a major involvement of PKC in the ddC-induced nociceptive behaviour. An increased expression of GAP43, a marker of neuroregeneration, and decreased levels of ATF3, a marker of neuroregeneration, were detected in all brain areas. ddC treatment also increased the expression of HuD, a RNA-binding protein target of PKC known to stabilize GAP43 mRNA. Pharmacological blockade of PKC prevented HuD and GAP43 overexpression. Silencing of both PKCγ and HuD reduced GAP43 levels in control mice and prevented the ddC-induced GAP43 enhanced expression. Present findings illustrate the presence of a supraspinal PKC-mediated HuD-GAP43 pathway activated by ddC. Based on our results, we speculate that antiretroviral drugs may recruit the HuD-GAP43 pathway, potentially contributing to a response to the antiretroviral neuronal toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mecp2 Mediates Experience-Dependent Transcriptional Upregulation of Ryanodine Receptor Type-3

PubMed Central

Torres, Rodrigo F.; Hidalgo, Cecilia; Kerr, Bredford

2017-01-01

Mecp2 is a DNA methylation reader that plays a critical role in experience-dependent plasticity. Increasing evidence supports a role for epigenetic modifications in activity-induced gene expression. Hence, candidate genes related to such phenomena are of great interest. Ryanodine receptors are intracellular calcium channels that contribute to hippocampal synaptic plasticity, dendritic spine remodeling, and participate in learning and memory processes. Here we exposed mice to the enriched environment (EE) paradigm, which through increased stimulation induces experience dependent-plasticity, to explore a role for methyl-cytosines, and Mecp2 in directing Ryanodine receptor 3 (Ryr3) transcriptional activity. EE induced a hippocampal-specific increase in the methylation of discrete cytosines located at a Ryr3 isoform promoter; chromatin immunoprecipitation experiments revealed that EE increased Mecp2 binding to this Ryr3 isoform promoter. Interestingly, the experimental paradigm induced robust Ryr3 upregulation, accompanied by miR132-dependent suppression of p250GAP, a pathway driving synaptogenesis. In contrast to WT mice, Mecp2-null mice showed diminished levels of Ryr3 and displayed impaired EE-induced Ryr3 upregulation, compromising miR132 dependent suppression of p250GAP and experience-dependent structural plasticity. Based on these results, we propose that Mecp2 acts as a transcriptional activator of Ryr3, contributing to experience-dependent plasticity. PMID:28659760

The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.

PubMed

Allen, Marcus J; Murphey, R K

2007-07-01

The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B(2) mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B(2) flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature-sensitive allele of the choline acetyltransferase gene (cha(ts2)) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B(2) flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function.

The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter

PubMed Central

Allen, Marcus J; Murphey, R K

2007-01-01

The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B2 mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B2 flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature-sensitive allele of the choline acetyltransferase gene (chats2) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B2 flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function. PMID:17650116

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Discovery and validation of a glioblastoma co-expressed gene module

PubMed Central

Dunwoodie, Leland J.; Poehlman, William L.; Ficklin, Stephen P.; Feltus, Frank Alexander

2018-01-01

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network. PMID:29541392

Discovery and validation of a glioblastoma co-expressed gene module.

PubMed

Dunwoodie, Leland J; Poehlman, William L; Ficklin, Stephen P; Feltus, Frank Alexander

2018-02-16

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages.

PubMed

Laird, Angela S; Mackovski, Nikolce; Rinkwitz, Silke; Becker, Thomas S; Giacomotto, Jean

2016-05-01

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

PubMed

Hu, Di; Zou, Hui; Han, Tao; Xie, Junze; Dai, Nannan; Zhuo, Liling; Gu, Jianhong; Bian, Jianchun; Yuan, Yan; Liu, Xuezhong; Liu, Zongping

2016-03-01

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca(2+) concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

PubMed

Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

2015-09-01

Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gender-Specific Gene Expression in Post-Mortem Human Brain: Localization to Sex Chromosomes

PubMed Central

Vawter, Marquis P; Evans, Simon; Choudary, Prabhakara; Tomita, Hiroaki; Meador-Woodruff, Jim; Molnar, Margherita; Li, Jun; Lopez, Juan F; Myers, Rick; Cox, David; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E

2011-01-01

Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex- chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. PMID:14583743

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

PubMed

Mishra, Sonal; Shukla, Aparna; Upadhyay, Swati; Sanchita; Sharma, Pooja; Singh, Seema; Phukan, Ujjal J; Meena, Abha; Khan, Feroz; Tripathi, Vineeta; Shukla, Rakesh Kumar; Shrama, Ashok

2014-04-01

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition. © 2013 Institute of Botany, Chinese Academy of Sciences.

Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids

PubMed Central

2011-01-01

Background Orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. In addition to the ecological significance, Phalaenopsis has been considered as an economically important floriculture industry worldwide. We aimed to use massively parallel 454 pyrosequencing for a global characterization of the Phalaenopsis transcriptome. Results To maximize sequence diversity, we pooled RNA from 10 samples of different tissues, various developmental stages, and biotic- or abiotic-stressed plants. We obtained 206,960 expressed sequence tags (ESTs) with an average read length of 228 bp. These reads were assembled into 8,233 contigs and 34,630 singletons. The unigenes were searched against the NCBI non-redundant (NR) protein database. Based on sequence similarity with known proteins, these analyses identified 22,234 different genes (E-value cutoff, e-7). Assembled sequences were annotated with Gene Ontology, Gene Family and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Among these annotations, over 780 unigenes encoding putative transcription factors were identified. Conclusion Pyrosequencing was effective in identifying a large set of unigenes from Phalaenopsis. The informative EST dataset we developed constitutes a much-needed resource for discovery of genes involved in various biological processes in Phalaenopsis and other orchid species. These transcribed sequences will narrow the gap between study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies. PMID:21749684

Gap junction-dependent homolog avoidance in the developing CNS.

PubMed

Baker, Michael W; Yazdani, Neema; Macagno, Eduardo R

2013-10-16

Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

The RasGAP Gene, RASAL2, is a Tumor and Metastasis Suppressor

PubMed Central

McLaughlin, Sara Koenig; Olsen, Sarah Naomi; Dake, Benjamin; De Raedt, Thomas; Lim, Elgene; Bronson, Roderick Terry; Beroukhim, Rameen; Polyak, Kornelia; Brown, Myles; Kuperwasser, Charlotte; Cichowski, Karen

2013-01-01

SUMMARY RAS genes are commonly mutated in cancer; however, RAS mutations are rare in breast cancer, despite the fact that Ras and ERK are frequently hyperactivated. Here we report that the RasGAP gene, RASAL2, functions as a tumor and metastasis suppressor. RASAL2 is mutated or suppressed in human breast cancer and RASAL2 ablation promotes tumor growth, progression, and metastasis in mouse models. In human breast cancer RASAL2-loss is associated with metastatic disease, low RASAL2 levels correlate with recurrence of luminal B tumors, and RASAL2 ablation promotes metastasis of luminal mouse tumors. Additional data reveal a broader role for RASAL2 inactivation in other tumor-types. These studies highlight the expanding role of RasGAPs and reveal an alternative mechanism of activating Ras in cancer. PMID:24029233

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Gene expression variability in human hepatic drug metabolizing enzymes and transporters.

PubMed

Yang, Lun; Price, Elvin T; Chang, Ching-Wei; Li, Yan; Huang, Ying; Guo, Li-Wu; Guo, Yongli; Kaput, Jim; Shi, Leming; Ning, Baitang

2013-01-01

Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications.

Sequence-based Network Completion Reveals the Integrality of Missing Reactions in Metabolic Networks*

PubMed Central

Krumholz, Elias W.; Libourel, Igor G. L.

2015-01-01

Genome-scale metabolic models are central in connecting genotypes to metabolic phenotypes. However, even for well studied organisms, such as Escherichia coli, draft networks do not contain a complete biochemical network. Missing reactions are referred to as gaps. These gaps need to be filled to enable functional analysis, and gap-filling choices influence model predictions. To investigate whether functional networks existed where all gap-filling reactions were supported by sequence similarity to annotated enzymes, four draft networks were supplemented with all reactions from the Model SEED database for which minimal sequence similarity was found in their genomes. Quadratic programming revealed that the number of reactions that could partake in a gap-filling solution was vast: 3,270 in the case of E. coli, where 72% of the metabolites in the draft network could connect a gap-filling solution. Nonetheless, no network could be completed without the inclusion of orphaned enzymes, suggesting that parts of the biochemistry integral to biomass precursor formation are uncharacterized. However, many gap-filling reactions were well determined, and the resulting networks showed improved prediction of gene essentiality compared with networks generated through canonical gap filling. In addition, gene essentiality predictions that were sensitive to poorly determined gap-filling reactions were of poor quality, suggesting that damage to the network structure resulting from the inclusion of erroneous gap-filling reactions may be predictable. PMID:26041773

Abstinence from cocaine-self-administration activates the nELAV/GAP -43 pathway in the hippocampus: A stress-related effect?

PubMed

Pascale, Alessia; Osera, Cecilia; Moro, Federico; Di Clemente, Angelo; Giannotti, Giuseppe; Caffino, Lucia; Govoni, Stefano; Fumagalli, Fabio; Cervo, Luigi

2016-06-01

We previously demonstrated that nELAV/GAP-43 pathway is pivotal for learning and its hippocampal expression is up-regulated by acute stress following repeated cocaine administration. We therefore hypothesized that abstinence-induced stress may sustain nELAV/GAP-43 pathway during early abstinence following 2 weeks of cocaine self-administration. We found that contingent, but not non-contingent, cocaine exposure selectively increases hippocampal nELAV, but not GAP-43, expression immediately after the last self-administration session, an effect that wanes after 24 h and that comes back 7 days later when nELAV activation becomes associated with increased expression of GAP-43, an effect again observed only in animals self-administering the psychostimulant. Such effect is specific for nELAV since the ubiquitous ELAV/HuR is unchanged. This nELAV profile suggests that its initial transient alteration is perhaps related to the daily administration of cocaine, while the increase in the nELAV/GAP-43 pathway following a week of abstinence may reflect the activation of this cascade as a target of stressful conditions associated with drug-related memories. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Gap junction disorders of myelinating cells.

PubMed

Kleopa, Kleopas A; Orthmann-Murphy, Jennifer; Sargiannidou, Irene

2010-01-01

Gap junctions (GJs) are channels that allow the diffusion of ions and small molecules across apposed cell membranes. In peripheral nerves, Schwann cells express the GJ proteins connexin32 (Cx32) and Cx29, which have distinct localizations. Cx32 forms GJs through non-compact myelin areas, whereas Cx29 forms hemichannels in the innermost layers of myelin apposing axonal Shaker-type K+ channels. In the CNS, rodent oligodendrocytes express Cx47, Cx32 and Cx29. Cx47 is expressed by all types of oligodendrocytes both in the white and grey matter and forms GJs on cell bodies and proximal processes, as well as most of the intercellular channels with astrocytes. Cx32 is expressed mostly by white matter oligodendrocytes and is localized in the myelin sheath of large diameter fibers. Cx29, and its human ortholog Cx31.3, appear to be restricted to oligodendrocytes that myelinate small caliber fibers, likely forming hemichannels. The importance of intercellular and intracellular GJs in myelinating cells are demonstrated by human disorders resulting from mutations affecting GJ proteins. The X-linked Charcot Marie Tooth disease (CMT1X) is caused by hundreds of mutations affecting Cx32. Patients with CMT1X present mainly with a progressive peripheral neuropathy, which may be accompanied by CNS myelin dysfunction. Mutations in Cx47 may cause a devastating leukodystrophy called Pelizaeus-Merzbacher-like disease or a milder spastic paraplegia. In addition, CNS demyelination may be caused by defects in genes expressing astrocytic GJ proteins, which are essential for oligodendrocytes. Findings from in vitro and in vivo models of these disorders developed over the last decade indicate that most mutations cause loss of function and an inability of the mutant connexins to form functional GJs. Here we review the clinical, genetic, and neurobiological aspects of GJ disorders affecting the PNS and CNS myelinating cells.

Wnt-related SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers

PubMed Central

Codocedo, Juan F.; Montecinos-Oliva, Carla; Inestrosa, Nibaldo C.

2015-01-01

Wnt-5a is a synaptogenic factor that modulates glutamatergic synapses and generates neuroprotection against Aβ oligomers. It is known that Wnt-5a plays a key role in the adult nervous system and synaptic plasticity. Emerging evidence indicates that miRNAs are actively involved in the regulation of synaptic plasticity. Recently, we showed that Wnt-5a is able to control the expression of several miRNAs including miR-101b, which has been extensively studied in carcinogenesis. However, its role in brain is just beginning to be explored. That is why we aim to study the relationship between Wnt-5a and miRNAs in glutamatergic synapses. We performed in silico analysis which predicted that miR-101b may inhibit the expression of synaptic GTPase-Activating Protein (SynGAP1), a Ras GTPase-activating protein critical for the development of cognition and proper synaptic function. Through overexpression of miR-101b, we showed that miR-101b is able to regulate the expression of SynGAP1 in an hippocampal cell line. Moreover and consistent with a decrease of miR-101b, Wnt-5a enhances SynGAP expression in cultured hippocampal neurons. Additionally, Wnt-5a increases the activity of SynGAP in a time-dependent manner, with a similar kinetic to CaMKII phosphorylation. This also, correlates with a modulation in the SynGAP clusters density. On the other hand, Aβ oligomers permanently decrease the number of SynGAP clusters. Interestingly, when neurons are co-incubated with Wnt-5a and Aβ oligomers, we do not observe the detrimental effect of Aβ oligomers, indicating that, Wnt-5a protects neurons from the synaptic failure triggered by Aβ oligomers. Overall, our findings suggest that SynGAP1 is part of the signaling pathways induced by Wnt-5a. Therefore, possibility exists that SynGAP is involved in the synaptic protection against Aβ oligomers. PMID:26124704

Constitutive expression of Botrytis aclada laccase in Pichia pastoris

PubMed Central

Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

2012-01-01

The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h). PMID:22705842

Accelerated Evolution of Developmentally Biased Genes in the Tetraphenic Ant Cardiocondyla obscurior.

PubMed

Schrader, Lukas; Helanterä, Heikki; Oettler, Jan

2017-03-01

Plastic gene expression underlies phenotypic plasticity and plastically expressed genes evolve under different selection regimes compared with ubiquitously expressed genes. Social insects are well-suited models to elucidate the evolutionary dynamics of plastic genes for their genetically and environmentally induced discrete polymorphisms. Here, we study the evolution of plastically expressed genes in the ant Cardiocondyla obscurior-a species that produces two discrete male morphs in addition to the typical female polymorphism of workers and queens. Based on individual-level gene expression data from 28 early third instar larvae, we test whether the same evolutionary dynamics that pertain to plastically expressed genes in adults also pertain to genes with plastic expression during development. In order to quantify plasticity of gene expression over multiple contrasts, we develop a novel geometric measure. For genes expressed during development, we show that plasticity of expression is positively correlated with evolutionary rates. We furthermore find a strong correlation between expression plasticity and expression variation within morphs, suggesting a close link between active and passive plasticity of gene expression. Our results support the notion of relaxed selection and neutral processes as important drivers in the evolution of adaptive plasticity. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Natural variation in non-coding regions underlying phenotypic diversity in budding yeast

PubMed Central

Salinas, Francisco; de Boer, Carl G.; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F.; Martínez, Claudio; Cubillos, Francisco A.

2016-01-01

Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype. PMID:26898953

A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

PubMed

Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

2013-09-01

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth. © 2013 International Society for Neurochemistry.

Global Transcriptome and Deletome Profiles of Yeast Exposed to Transition Metals

PubMed Central

Jin, Yong Hwan; Dunlap, Paul E.; McBride, Sandra J.; Al-Refai, Hanan; Bushel, Pierre R.; Freedman, Jonathan H.

2008-01-01

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways. PMID:18437200

A transcriptional blueprint for a spiral-cleaving embryo.

PubMed

Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

2016-08-05

The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

MAP kinase pathways in the yeast Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

1998-01-01

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Integrated metabolic flux and omics analysis of Synechocystis sp. PCC 6803 under mixotrophic and photoheterotrophic conditions.

PubMed

Nakajima, Tsubasa; Kajihata, Shuichi; Yoshikawa, Katsunori; Matsuda, Fumio; Furusawa, Chikara; Hirasawa, Takashi; Shimizu, Hiroshi

2014-09-01

Cyanobacteria have flexible metabolic capability that enables them to adapt to various environments. To investigate their underlying metabolic regulation mechanisms, we performed an integrated analysis of metabolic flux using transcriptomic and metabolomic data of a cyanobacterium Synechocystis sp. PCC 6803, under mixotrophic and photoheterotrophic conditions. The integrated analysis indicated drastic metabolic flux changes, with much smaller changes in gene expression levels and metabolite concentrations between the conditions, suggesting that the flux change was not caused mainly by the expression levels of the corresponding genes. Under photoheterotrophic conditions, created by the addition of the photosynthesis inhibitor atrazine in mixotrophic conditions, the result of metabolic flux analysis indicated the significant repression of carbon fixation and the activation of the oxidative pentose phosphate pathway (PPP). Moreover, we observed gluconeogenic activity of upstream of glycolysis, which enhanced the flux of the oxidative PPP to compensate for NADPH depletion due to the inhibition of the light reaction of photosynthesis. 'Omics' data suggested that these changes were probably caused by the repression of the gap1 gene, which functions as a control valve in the metabolic network. Since metabolic flux is the outcome of a complicated interplay of cellular components, integrating metabolic flux with other 'omics' layers can identify metabolic changes and narrow down these regulatory mechanisms more effectively. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

PubMed

Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

2017-01-15

In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. The transfection of PAMS/DNA/10NLS had less dependence on the breakdown of nuclear envelope. Both the nuclear import and transfection efficiency of PAMS/DNA/10NLS were higher in RanGAP1 overexpressed cells than that in normal cells. Moreover, the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Analysis of multiplex gene expression maps obtained by voxelation.

PubMed

An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

2009-04-29

Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental results confirm the hypothesis that genes with similar gene expression maps might have similar gene functions. The voxelation data takes into account the location information of gene expression level in mouse brain, which is novel in related research. The proposed approach can potentially be used to predict gene functions and provide helpful suggestions to biologists.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

PubMed Central

Ning, ZhongHua; Hincke, Maxwell T.; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences. PMID:24676480

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

PubMed

Zhang, Quan; Liu, Long; Zhu, Feng; Ning, ZhongHua; Hincke, Maxwell T; Yang, Ning; Hou, ZhuoCheng

2014-01-01

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Co-expression network analysis of duplicate genes in maize (Zea mays L.) reveals no subgenome bias.

PubMed

Li, Lin; Briskine, Roman; Schaefer, Robert; Schnable, Patrick S; Myers, Chad L; Flagel, Lex E; Springer, Nathan M; Muehlbauer, Gary J

2016-11-04

Gene duplication is prevalent in many species and can result in coding and regulatory divergence. Gene duplications can be classified as whole genome duplication (WGD), tandem and inserted (non-syntenic). In maize, WGD resulted in the subgenomes maize1 and maize2, of which maize1 is considered the dominant subgenome. However, the landscape of co-expression network divergence of duplicate genes in maize is still largely uncharacterized. To address the consequence of gene duplication on co-expression network divergence, we developed a gene co-expression network from RNA-seq data derived from 64 different tissues/stages of the maize reference inbred-B73. WGD, tandem and inserted gene duplications exhibited distinct regulatory divergence. Inserted duplicate genes were more likely to be singletons in the co-expression networks, while WGD duplicate genes were likely to be co-expressed with other genes. Tandem duplicate genes were enriched in the co-expression pattern where co-expressed genes were nearly identical for the duplicates in the network. Older gene duplications exhibit more extensive co-expression variation than younger duplications. Overall, non-syntenic genes primarily from inserted duplications show more co-expression divergence. Also, such enlarged co-expression divergence is significantly related to duplication age. Moreover, subgenome dominance was not observed in the co-expression networks - maize1 and maize2 exhibit similar levels of intra subgenome correlations. Intriguingly, the level of inter subgenome co-expression was similar to the level of intra subgenome correlations, and genes from specific subgenomes were not likely to be the enriched in co-expression network modules and the hub genes were not predominantly from any specific subgenomes in maize. Our work provides a comprehensive analysis of maize co-expression network divergence for three different types of gene duplications and identifies potential relationships between duplication types, duplication ages and co-expression consequences.

Berberine potentizes apoptosis induced by X-rays irradiation probably through modulation of gap junctions.

PubMed

Liu, Bing; Wang, Qin; Yuan, Dong-dong; Hong, Xiao-ting; Tao, Liang

2011-04-01

Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this effect and gap junction intercellular communication (GJIC). The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by manipulation of connexin (Cx) expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine. In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only in the presence of functional gap junctions. These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably through enhancement of gap junction activity.

Tissue Non-Specific Genes and Pathways Associated with Diabetes: An Expression Meta-Analysis.

PubMed

Mei, Hao; Li, Lianna; Liu, Shijian; Jiang, Fan; Griswold, Michael; Mosley, Thomas

2017-01-21

We performed expression studies to identify tissue non-specific genes and pathways of diabetes by meta-analysis. We searched curated datasets of the Gene Expression Omnibus (GEO) database and identified 13 and five expression studies of diabetes and insulin responses at various tissues, respectively. We tested differential gene expression by empirical Bayes-based linear method and investigated gene set expression association by knowledge-based enrichment analysis. Meta-analysis by different methods was applied to identify tissue non-specific genes and gene sets. We also proposed pathway mapping analysis to infer functions of the identified gene sets, and correlation and independent analysis to evaluate expression association profile of genes and gene sets between studies and tissues. Our analysis showed that PGRMC1 and HADH genes were significant over diabetes studies, while IRS1 and MPST genes were significant over insulin response studies, and joint analysis showed that HADH and MPST genes were significant over all combined data sets. The pathway analysis identified six significant gene sets over all studies. The KEGG pathway mapping indicated that the significant gene sets are related to diabetes pathogenesis. The results also presented that 12.8% and 59.0% pairwise studies had significantly correlated expression association for genes and gene sets, respectively; moreover, 12.8% pairwise studies had independent expression association for genes, but no studies were observed significantly different for expression association of gene sets. Our analysis indicated that there are both tissue specific and non-specific genes and pathways associated with diabetes pathogenesis. Compared to the gene expression, pathway association tends to be tissue non-specific, and a common pathway influencing diabetes development is activated through different genes at different tissues.

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

PubMed

Berman, Joan W; Carvallo, Loreto; Buckner, Clarisa M; Luers, Aimée; Prevedel, Lisa; Bennett, Michael V; Eugenin, Eliseo A

2016-03-02

HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.

PubMed Central

Bookstein, R; Lee, E Y; To, H; Young, L J; Sery, T W; Hayes, R C; Friedmann, T; Lee, W H

1988-01-01

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families. Images PMID:2895471

Studies of Neurofibromatosis-1 Modifier Genes

DTIC Science & Technology

2005-06-01

inhibitors of GTPase activation by preventing the dissociation of GDP from the inactive GTPase. [E3 DDff The current dogma, at least in the context of...dissociation the cycle is less clear. inhibitors (GD/s), the activity of each of which is potentially modulated in response to various signals. Inactive...function No. of No. of No. of No. of No. of ArfGAPs RabGAPs RapGAPs RasGAPs RhoGAPs BAR IPR004148 Membrane curvature sensor 6 (4) 0 0 0 6 (6) BTK

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

NASA Technical Reports Server (NTRS)

Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

2003-01-01

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

PubMed

Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

2015-12-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

PubMed Central

Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

2015-01-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution. PMID:26685068

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

General statistics of stochastic process of gene expression in eukaryotic cells.

PubMed Central

Kuznetsov, V A; Knott, G D; Bonner, R F

2002-01-01

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations. PMID:12136033

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

PubMed

Liu, Shuo; Niger, Corinne; Koh, Eugene Y; Stains, Joseph P

2015-01-01

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.

The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.

PubMed

Wang, Ping; Li, Yong; Nie, Huiqiong; Zhang, Xiaoyan; Shao, Qiongyan; Hou, Xiuli; Xu, Wen; Hong, Weisong; Xu, Aie

2016-10-01

Vitiligo is a common acquired depigmentation skin disease characterized by loss or dysfunction of melanocytes within the skin lesion, but its pathologenesis is far from lucid. The gene expression profiling of segmental vitiligo (SV) and generalized vitiligo (GV) need further investigation. To better understanding the common and distinct factors, especially in the view of gene expression profile, which were involved in the diseases development and maintenance of segmental vitiligo (SV) and generalized vitiligo (GV). Peripheral bloods were collected from SV, GV and healthy individual (HI), followed by leukocytes separation and total RNA extraction. The high-throughput whole genome expression microarrays were used to assay the gene expression profiles between HI, SV and GV. Bioinformatics tools were employed to annotated the biological function of differently expressed genes. Quantitative PCR assay was used to validate the gene expression of array. Compared to HI, 239 over-expressed genes and 175 down-expressed genes detected in SV, 688 over-expressed genes and 560 down-expressed genes were found in GV, following the criteria of log2 (fold change)≥0.585 and P value<0.05. In these differently expressed genes, 60 over-expressed genes and 60 down-expressed genes had similar tendency in SV and GV. Compared to SV, 223 genes were up regulated and 129 genes were down regulated in GV. In the SV with HI as control, the differently expressed genes were mainly involved in the adaptive immune response, cytokine-cytokine receptor interaction, chemokine signaling, focal adhesion and sphingolipid metabolism. The differently expressed genes between GV and HI were mainly involved in the innate immune, autophagy, apoptosis, melanocyte biology, ubiquitin mediated proteolysis and tyrosine metabolism, which was different from SV. While the differently expressed genes between SV and GV were mainly involved in the metabolism pathway of purine, pyrimidine, glycolysis and sphingolipid. Above results suggested that they not only shared part bio-process and signal pathway, but more important, they utilized different biological mechanism in their pathogenesis and maintenance. Our results provide a comprehensive view on the gene expression profiling change between SV and GV especially in the side of leukocytes, and may facilitate the future study on their molecular mechanism and theraputic targets. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Vitamin C Transporters in Cancer: Current Understanding and Gaps in Knowledge

PubMed Central

Wohlrab, Christina; Phillips, Elisabeth; Dachs, Gabi U.

2017-01-01

Sufficient uptake and whole body distribution of vitamin C (ascorbate) is essential for many biochemical processes, including some that are vital for tumor growth and spread. Uptake of ascorbate into cancer cells is modulated by availability, tumor blood flow, tissue diffusion parameters, and ascorbate transport proteins. Uptake into cells is mediated by two families of transport proteins, namely, the solute carrier gene family 23, consisting of sodium-dependent vitamin C transporters (SVCTs) 1 and 2, and the SLC2 family of glucose transporters (GLUTs). GLUTs transport the oxidized form of the vitamin, dehydroascorbate (DHA), which is present at negligible to low physiological levels. SVCT1 and 2 are capable of accumulating ascorbate against a concentration gradient from micromolar concentrations outside to millimolar levels inside of cells. Investigating the expression and regulation of SVCTs in cancer has only recently started to be included in studies focused on the role of ascorbate in tumor formation, progression, and response to therapy. This review gives an overview of the current, limited knowledge of ascorbate transport across membranes, as well as tissue distribution, gene expression, and the relevance of SVCTs in cancer. As tumor ascorbate accumulation may play a role in the anticancer activity of high dose ascorbate treatment, further research into ascorbate transport in cancer tissue is vital. PMID:28484682

In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.

PubMed

Law, Lee Yong; Zhang, Wei V; Stott, N Susan; Becker, David L; Green, Colin R

2006-09-01

The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

PubMed Central

Højland, Dorte H.; Jensen, Karl-Martin Vagn; Kristensen, Michael

2014-01-01

Background The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain. Results We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F1, F13 and F29 to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F29 than in F1. For males, about half of the genes accessed in the global gene expression were up-regulated in F13 and F29 in comparison with the F1 population. In females, 60% of the genes were up-regulated in F13 in comparison with F1, while 33% were up-regulated in F29. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F13 flies in comparison with F1. Gene expression then increased from F13 to F29 in males and decreased further in females. Conclusion The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains. PMID:24489682

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

PubMed

Ma, Ke-Tao; Li, Xin-Zhi; Li, Li; Jiang, Xue-Wei; Chen, Xin-Yan; Liu, Wei-Dong; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang

2014-02-01

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

An RNA-Seq based gene expression atlas of the common bean.

PubMed

O'Rourke, Jamie A; Iniguez, Luis P; Fu, Fengli; Bucciarelli, Bruna; Miller, Susan S; Jackson, Scott A; McClean, Philip E; Li, Jun; Dai, Xinbin; Zhao, Patrick X; Hernandez, Georgina; Vance, Carroll P

2014-10-06

Common bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species. Combining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form. These data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

Sequence-based Network Completion Reveals the Integrality of Missing Reactions in Metabolic Networks.

PubMed

Krumholz, Elias W; Libourel, Igor G L

2015-07-31

Genome-scale metabolic models are central in connecting genotypes to metabolic phenotypes. However, even for well studied organisms, such as Escherichia coli, draft networks do not contain a complete biochemical network. Missing reactions are referred to as gaps. These gaps need to be filled to enable functional analysis, and gap-filling choices influence model predictions. To investigate whether functional networks existed where all gap-filling reactions were supported by sequence similarity to annotated enzymes, four draft networks were supplemented with all reactions from the Model SEED database for which minimal sequence similarity was found in their genomes. Quadratic programming revealed that the number of reactions that could partake in a gap-filling solution was vast: 3,270 in the case of E. coli, where 72% of the metabolites in the draft network could connect a gap-filling solution. Nonetheless, no network could be completed without the inclusion of orphaned enzymes, suggesting that parts of the biochemistry integral to biomass precursor formation are uncharacterized. However, many gap-filling reactions were well determined, and the resulting networks showed improved prediction of gene essentiality compared with networks generated through canonical gap filling. In addition, gene essentiality predictions that were sensitive to poorly determined gap-filling reactions were of poor quality, suggesting that damage to the network structure resulting from the inclusion of erroneous gap-filling reactions may be predictable. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1.

PubMed

Chu, Miensheng; Novak, Stefanie Mares; Cover, Cathleen; Wang, Anne A; Chinyere, Ikeotunye Royal; Juneman, Elizabeth B; Zarnescu, Daniela C; Wong, Pak Kin; Gregorio, Carol C

2018-02-06

Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM. In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart. © 2017 American Heart Association, Inc.

Analysis of bHLH coding genes using gene co-expression network approach.

PubMed

Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

2016-07-01

Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species.

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Characterization of basal gene expression trends over a diurnal cycle in Xiphophorus maculatus skin, brain and liver.

PubMed

Lu, Yuan; Reyes, Jose; Walter, Sean; Gonzalez, Trevor; Medrano, Geraldo; Boswell, Mikki; Boswell, William; Savage, Markita; Walter, Ronald

2018-06-01

Evolutionarily conserved diurnal circadian mechanisms maintain oscillating patterns of gene expression based on the day-night cycle. Xiphophorus fish have been used to evaluate transcriptional responses after exposure to various light sources and it was determined that each source incites distinct genetic responses in skin tissue. However, basal expression levels of genes that show oscillating expression patterns in day-night cycle, may affect the outcomes of such experiments, since basal gene expression levels at each point in the circadian path may influence the profile of identified light responsive genes. Lack of knowledge regarding diurnal fluctuations in basal gene expression patterns may confound the understanding of genetic responses to external stimuli (e.g., light) since the dynamic nature of gene expression implies animals subjected to stimuli at different times may be at very different stages within the continuum of genetic homeostasis. We assessed basal gene expression changes over a 24-hour period in 200 select Xiphophorus gene targets known to transcriptionally respond to various types of light exposure. We identified 22 genes in skin, 36 genes in brain and 28 genes in liver that exhibit basal oscillation of expression patterns. These genes, including known circadian regulators, produced the expected expression patterns over a 24-hour cycle when compared to circadian regulatory genes identified in other species, especially human and other vertebrate animal models. Our results suggest the regulatory network governing diurnal oscillating gene expression is similar between Xiphophorus and other vertebrates for the three Xiphophorus organs tested. In addition, we were able to categorize light responsive gene sets in Xiphophorus that do, and do not, exhibit circadian based oscillating expression patterns. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Life-cycle and growth-phase-dependent regulation of the ubiquitin genes of Trypanosoma cruzi.

PubMed

Manning-Cela, Rebeca; Jaishankar, Sobha; Swindle, John

2006-07-01

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Although the level of CAT activity in logarithmic growing epimastigotes is six- to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia

PubMed Central

Alves, Cecília J.; Leitão, Luís; Sousa, Daniela M.; Alencastre, Inês S.; Conceição, Francisco; Lamghari, Meriem

2017-01-01

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system. PMID:28742111

Gene expression changes with age in skin, adipose tissue, blood and brain.

PubMed

Glass, Daniel; Viñuela, Ana; Davies, Matthew N; Ramasamy, Adaikalavan; Parts, Leopold; Knowles, David; Brown, Andrew A; Hedman, Asa K; Small, Kerrin S; Buil, Alfonso; Grundberg, Elin; Nica, Alexandra C; Di Meglio, Paola; Nestle, Frank O; Ryten, Mina; Durbin, Richard; McCarthy, Mark I; Deloukas, Panagiotis; Dermitzakis, Emmanouil T; Weale, Michael E; Bataille, Veronique; Spector, Tim D

2013-07-26

Previous studies have demonstrated that gene expression levels change with age. These changes are hypothesized to influence the aging rate of an individual. We analyzed gene expression changes with age in abdominal skin, subcutaneous adipose tissue and lymphoblastoid cell lines in 856 female twins in the age range of 39-85 years. Additionally, we investigated genotypic variants involved in genotype-by-age interactions to understand how the genomic regulation of gene expression alters with age. Using a linear mixed model, differential expression with age was identified in 1,672 genes in skin and 188 genes in adipose tissue. Only two genes expressed in lymphoblastoid cell lines showed significant changes with age. Genes significantly regulated by age were compared with expression profiles in 10 brain regions from 100 postmortem brains aged 16 to 83 years. We identified only one age-related gene common to the three tissues. There were 12 genes that showed differential expression with age in both skin and brain tissue and three common to adipose and brain tissues. Skin showed the most age-related gene expression changes of all the tissues investigated, with many of the genes being previously implicated in fatty acid metabolism, mitochondrial activity, cancer and splicing. A significant proportion of age-related changes in gene expression appear to be tissue-specific with only a few genes sharing an age effect in expression across tissues. More research is needed to improve our understanding of the genetic influences on aging and the relationship with age-related diseases.

Differential expression profiles and pathways of genes in sugarcane leaf at elongation stage in response to drought stress

PubMed Central

Li, Changning; Nong, Qian; Solanki, Manoj Kumar; Liang, Qiang; Xie, Jinlan; Liu, Xiaoyan; Li, Yijie; Wang, Weizan; Yang, Litao; Li, Yangrui

2016-01-01

Water stress causes considerable yield losses in sugarcane. To investigate differentially expressed genes under water stress, a pot experiment was performed with the sugarcane variety GT21 at three water-deficit levels (mild, moderate, and severe) during the elongation stage and gene expression was analyzed using microarray technology. Physiological parameters of sugarcane showed significant alterations in response to drought stress. Based on the expression profile of 15,593 sugarcane genes, 1,501 (9.6%) genes were differentially expressed under different water-level treatments; 821 genes were upregulated and 680 genes were downregulated. A gene similarity analysis showed that approximately 62.6% of the differentially expressed genes shared homology with functional proteins. In a Gene Ontology (GO) analysis, 901 differentially expressed genes were assigned to 36 GO categories. Moreover, 325 differentially expressed genes were classified into 101 pathway categories involved in various processes, such as the biosynthesis of secondary metabolites, ribosomes, carbon metabolism, etc. In addition, some unannotated genes were detected; these may provide a basis for studies of water-deficit tolerance. The reliability of the observed expression patterns was confirmed by RT-PCR. The results of this study may help identify useful genes for improving drought tolerance in sugarcane. PMID:27170459

The JAK-STAT signaling pathway: input and output integration.

PubMed

Murray, Peter J

2007-03-01

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.

Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes.

PubMed

Le Berre, Maël; Aubertin, Johannes; Piel, Matthieu

2012-11-01

The quest to understand how the mechanical and geometrical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical and simple cell culture plate and the biological reality of actual tissue. In tissues, cells have their physical space constrained by neighboring cells and the extracellular matrix. Here, we propose a simple and versatile device to precisely and dynamically control this confinement parameter in cultured cells. We show that there is a precise threshold deformation above which the nuclear lamina breaks and reconstructs, whereas nuclear volume changes. We also show that different nuclear deformations correlate with the expression of specific sets of genes, including nuclear factors and classical mechanotransduction pathways. This versatile device thus enables the precise control of cell and nuclear deformation by confinement and the correlative study of the associated molecular events.

MorphoGraphX: A platform for quantifying morphogenesis in 4D.

PubMed

Barbier de Reuille, Pierre; Routier-Kierzkowska, Anne-Lise; Kierzkowski, Daniel; Bassel, George W; Schüpbach, Thierry; Tauriello, Gerardo; Bajpai, Namrata; Strauss, Sören; Weber, Alain; Kiss, Annamaria; Burian, Agata; Hofhuis, Hugo; Sapala, Aleksandra; Lipowczan, Marcin; Heimlicher, Maria B; Robinson, Sarah; Bayer, Emmanuelle M; Basler, Konrad; Koumoutsakos, Petros; Roeder, Adrienne H K; Aegerter-Wilmsen, Tinri; Nakayama, Naomi; Tsiantis, Miltos; Hay, Angela; Kwiatkowska, Dorota; Xenarios, Ioannis; Kuhlemeier, Cris; Smith, Richard S

2015-05-06

Morphogenesis emerges from complex multiscale interactions between genetic and mechanical processes. To understand these processes, the evolution of cell shape, proliferation and gene expression must be quantified. This quantification is usually performed either in full 3D, which is computationally expensive and technically challenging, or on 2D planar projections, which introduces geometrical artifacts on highly curved organs. Here we present MorphoGraphX ( www.MorphoGraphX.org), a software that bridges this gap by working directly with curved surface images extracted from 3D data. In addition to traditional 3D image analysis, we have developed algorithms to operate on curved surfaces, such as cell segmentation, lineage tracking and fluorescence signal quantification. The software's modular design makes it easy to include existing libraries, or to implement new algorithms. Cell geometries extracted with MorphoGraphX can be exported and used as templates for simulation models, providing a powerful platform to investigate the interactions between shape, genes and growth.

GSEH: A Novel Approach to Select Prostate Cancer-Associated Genes Using Gene Expression Heterogeneity.

PubMed

Kim, Hyunjin; Choi, Sang-Min; Park, Sanghyun

2018-01-01

When a gene shows varying levels of expression among normal people but similar levels in disease patients or shows similar levels of expression among normal people but different levels in disease patients, we can assume that the gene is associated with the disease. By utilizing this gene expression heterogeneity, we can obtain additional information that abets discovery of disease-associated genes. In this study, we used collaborative filtering to calculate the degree of gene expression heterogeneity between classes and then scored the genes on the basis of the degree of gene expression heterogeneity to find "differentially predicted" genes. Through the proposed method, we discovered more prostate cancer-associated genes than 10 comparable methods. The genes prioritized by the proposed method are potentially significant to biological processes of a disease and can provide insight into them.

Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

PubMed

Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

2017-03-01

The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC in the future. ii) Differentially expressed genes may be involved in the occurrence of GC via a variety of mechanisms. iii) CD44, CXXC5, MYH9, MALAT1 and other genes may have important implications for the occurrence and development of GC through the regulation, interaction, and mutual influence of circRNA-miRNA-mRNA via different mechanisms.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-01-01

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

Genetic and epigenetic architecture of sex-biased expression in the jewel wasps Nasonia vitripennis and giraulti

PubMed Central

Wang, Xu; Werren, John H.; Clark, Andrew G.

2015-01-01

There is extraordinary diversity in sexual dimorphism (SD) among animals, but little is known about its epigenetic basis. To study the epigenetic architecture of SD in a haplodiploid system, we performed RNA-seq and whole-genome bisulfite sequencing of adult females and males from two closely related parasitoid wasps, Nasonia vitripennis and Nasonia giraulti. More than 75% of expressed genes displayed significantly sex-biased expression. As a consequence, expression profiles are more similar between species within each sex than between sexes within each species. Furthermore, extremely male- and female-biased genes are enriched for totally different functional categories: male-biased genes for key enzymes in sex-pheromone synthesis and female-biased genes for genes involved in epigenetic regulation of gene expression. Remarkably, just 70 highly expressed, extremely male-biased genes account for 10% of all transcripts in adult males. Unlike expression profiles, DNA methylomes are highly similar between sexes within species, with no consistent sex differences in methylation found. Therefore, methylation changes cannot explain the extensive level of sex-biased gene expression observed. Female-biased genes have smaller sequence divergence between species, higher conservation to other hymenopterans, and a broader expression range across development. Overall, female-biased genes have been recruited from genes with more conserved and broadly expressing “house-keeping” functions, whereas male-biased genes are more recently evolved and are predominately testis specific. In summary, Nasonia accomplish a striking degree of sex-biased expression without sex chromosomes or epigenetic differences in methylation. We propose that methylation provides a general signal for constitutive gene expression, whereas other sex-specific signals cause sex-biased gene expression. PMID:26100871

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

PubMed

Van Gelder, R N; Bae, H; Palazzolo, M J; Krasnow, M A

1995-12-01

Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Polycistronic gene expression in Aspergillus niger.

PubMed

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

PubMed

Lin, Changpo; Tang, Xiao; Xu, Lirong; Qian, Ruizhe; Shi, Zhenyu; Wang, Lixin; Cai, Tingting; Yan, Dong; Fu, Weiguo; Guo, Daqiao

2017-07-10

The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

Double-filter identification of vascular-expressed genes using Arabidopsis plants with vascular hypertrophy and hypotrophy.

PubMed

Ckurshumova, Wenzislava; Scarpella, Enrico; Goldstein, Rochelle S; Berleth, Thomas

2011-08-01

Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

PubMed

Carpentier, Jean; Grenier, Eric; Esquibet, Magalie; Hamel, Louis-Philippe; Moffett, Peter; Manzanares-Dauleux, Maria J; Kerlan, Marie-Claire

2013-04-19

The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene appears to evolve under purifying selection. Its variability does not seem to influence the specificity of GPA2 recognition but is able to modulate this activity by enhancing the defence response. It seems therefore that the interaction with the plant resistance protein GPA2 (and/or Rx) rather than with the nematode effector was the major force in the evolution of the RanGAP2 locus in potato. From a mechanistic point of view these results are in accordance with a physical interaction of RanGAP2 with GPA2 and suggest that RBP-1 would rather bind the RanGAP2-GPA2 complex than the RanGAP2 protein alone.

Increased gene expression noise in human cancers is correlated with low p53 and immune activities as well as late stage cancer.

PubMed

Han, Rongfei; Huang, Guanqun; Wang, Yejun; Xu, Yafei; Hu, Yueming; Jiang, Wenqi; Wang, Tianfu; Xiao, Tian; Zheng, Duo

2016-11-01

Gene expression in metazoans is delicately organized. As genetic information transmits from DNA to RNA and protein, expression noise is inevitably generated. Recent studies begin to unveil the mechanisms of gene expression noise control, but the changes of gene expression precision in pathologic conditions like cancers are unknown. Here we analyzed the transcriptomic data of human breast, liver, lung and colon cancers, and found that the expression noise of more than 74.9% genes was increased in cancer tissues as compared to adjacent normal tissues. This suggested that gene expression precision controlling collapsed during cancer development. A set of 269 genes with noise increased more than 2-fold were identified across different cancer types. These genes were involved in cell adhesion, catalytic and metabolic functions, implying the vulnerability of deregulation of these processes in cancers. We also observed a tendency of increased expression noise in patients with low p53 and immune activity in breast, liver and lung caners but not in colon cancers, which indicated the contributions of p53 signaling and host immune surveillance to gene expression noise in cancers. Moreover, more than 53.7% genes had increased noise in patients with late stage than early stage cancers, suggesting that gene expression precision was associated with cancer outcome. Together, these results provided genomic scale explorations of gene expression noise control in human cancers.

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

PubMed

Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

2012-04-01

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Gene Architectures that Minimize Cost of Gene Expression.

PubMed

Frumkin, Idan; Schirman, Dvir; Rotman, Aviv; Li, Fangfei; Zahavi, Liron; Mordret, Ernest; Asraf, Omer; Wu, Song; Levy, Sasha F; Pilpel, Yitzhak

2017-01-05

Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

PubMed Central

2012-01-01

Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. Results We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. PMID:22853646

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

PubMed

Roy, Ankita; Goodman, Joshua H; Begum, Gulnaz; Donnelly, Bridget F; Pittman, Gabrielle; Weinman, Edward J; Sun, Dandan; Subramanya, Arohan R

2015-02-15

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

Functional clustering of time series gene expression data by Granger causality

PubMed Central

2012-01-01

Background A common approach for time series gene expression data analysis includes the clustering of genes with similar expression patterns throughout time. Clustered gene expression profiles point to the joint contribution of groups of genes to a particular cellular process. However, since genes belong to intricate networks, other features, besides comparable expression patterns, should provide additional information for the identification of functionally similar genes. Results In this study we perform gene clustering through the identification of Granger causality between and within sets of time series gene expression data. Granger causality is based on the idea that the cause of an event cannot come after its consequence. Conclusions This kind of analysis can be used as a complementary approach for functional clustering, wherein genes would be clustered not solely based on their expression similarity but on their topological proximity built according to the intensity of Granger causality among them. PMID:23107425

Spinal astrocyte gap junctions contribute to oxaliplatin-induced mechanical hypersensitivity.

PubMed

Yoon, Seo-Yeon; Robinson, Caleb R; Zhang, Haijun; Dougherty, Patrick M

2013-02-01

Spinal glial cells contribute to the development of many types of inflammatory and neuropathic pain. Here the contribution of spinal astrocytes and astrocyte gap junctions to oxaliplatin-induced mechanical hypersensitivity was explored. The expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn was significantly increased at day 7 but recovered at day 14 after oxaliplatin treatment, suggesting a transient activation of spinal astrocytes by chemotherapy. Astrocyte-specific gap junction protein connexin 43 (Cx43) was significantly increased in dorsal horn at both day 7 and day 14 following chemotherapy, but neuronal (connexin 36 [Cx36]) and oligodendrocyte (connexin 32 [Cx32]) gap junction proteins did not show any change. Blockade of astrocyte gap junction with carbenoxolone (CBX) prevented oxaliplatin-induced mechanical hypersensitivity in a dose-dependent manner and the increase of spinal GFAP expression, but had no effect once the mechanical hypersensitivity induced by oxaliplatin had fully developed. These results suggest that oxaliplatin chemotherapy induces the activation of spinal astrocytes and this is accompanied by increased expression of astrocyte-astrocyte gap junction connections via Cx43. These alterations in spinal astrocytes appear to contribute to the induction but not the maintenance of oxaliplatin-induced mechanical hypersensitivity. Combined, these results suggest that targeting spinal astrocyte/astrocyte-specific gap junction could be a new therapeutic strategy to prevent oxaliplatin-induced neuropathy. Spinal astrocytes but not microglia were recently shown to be recruited in paclitaxel-related chemoneuropathy. Here, spinal astrocyte gap junctions are shown to play an important role in the induction of oxaliplatin neuropathy. Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Intermediate introns in nuclear genes of euglenids - are they a distinct type?

PubMed

Milanowski, Rafał; Gumińska, Natalia; Karnkowska, Anna; Ishikawa, Takao; Zakryś, Bożena

2016-02-29

Nuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, -7 and -6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

PubMed

Li, Zibo; Heng, Jianfu; Yan, Jinhua; Guo, Xinwu; Tang, Lili; Chen, Ming; Peng, Limin; Wu, Yepeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Wang, Jun

2016-11-01

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum

PubMed Central

2013-01-01

Background METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. Methods We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Results Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Conclusion Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites. PMID:23937714

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum.

PubMed

Cadet, Jean Lud; Jayanthi, Subramaniam; McCoy, Michael T; Ladenheim, Bruce; Saint-Preux, Fabienne; Lehrmann, Elin; De, Supriyo; Becker, Kevin G; Brannock, Christie

2013-08-12

METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites.