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Sample records for gene disrupts development

  1. Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology.

    Science.gov (United States)

    Zheng, Yan-Mei; Lin, Fu-Long; Gao, Hao; Zou, Gen; Zhang, Jiang-Wei; Wang, Gao-Qian; Chen, Guo-Dong; Zhou, Zhi-Hua; Yao, Xin-Sheng; Hu, Dan

    2017-08-23

    Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-based system has become a common platform for genome editing in a variety of organisms. However, recent application of this technology in filamentous fungi is limited to model strains, a versatile method for efficient gene disruption in different fungi is lacking. Here, we investigated the utility of the CRISPR-Cas9 system in a less-studied fungus Nodulisporium sp. (No. 65-12-7-1), and we have developed an efficient CRISPR-Cas9-based gene disruption strategy by simultaneous transformation of in vitro transcriptional gRNA and the linear maker gene cassette into the Cas9-expressing fungi. We found that the linear marker gene cassette could not only allow for selection of transformants, but also significantly enhance the gene disruption efficiency by inserting itself into the Cas9 cut site. Moreover, the above approach also demonstrated its efficiency in two other phylogenetically distinct strains Aspergillus oryzae NSAR1 and Sporormiella minima (No. 40-1-4-1) from two different classes of Ascomycota. These results suggested that a versatile CRISPR-Cas9-based gene disruption method in filamentous fungi was established.

  2. A potential disruptive technology in vaccine development: gene-based vaccines and their application to infectious diseases.

    Science.gov (United States)

    Kaslow, David C

    2004-10-01

    Vaccine development requires an amalgamation of disparate disciplines and has unique economic and regulatory drivers. Non-viral gene-based delivery systems, such as formulated plasmid DNA, are new and potentially disruptive technologies capable of providing 'cheaper, simpler, and more convenient-to-use' vaccines. Typically and somewhat ironically, disruptive technologies have poorer product performance, at least in the near-term, compared with the existing conventional technologies. Because successful product development requires that the product's performance must meet or exceed the efficacy threshold for a desired application, the appropriate selection of the initial product applications for a disruptive technology is critical for its successful evolution. In this regard, the near-term successes of gene-based vaccines will likely be for protection against bacterial toxins and acute viral and bacterial infections. Recent breakthroughs, however, herald increasing rather than languishing performance improvements in the efficacy of gene-based vaccines. Whether gene-based vaccines ultimately succeed in eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria and tuberculosis, for which the conventional vaccine technologies have failed, remains to be determined. A success against any one of the persistent intracellular pathogens would be sufficient proof that gene-based vaccines represent a disruptive technology against which future vaccine technologies will be measured.

  3. The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development

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    Veldhoen, Nik [Department of Biochemistry and Microbiology, P.O. Box 3055, Stn. CSC, University of Victoria, Victoria, British Columbia V8W 3P6 (Canada); Skirrow, Rachel C. [Pacific Environmental Science Centre, 2645 Dollarton Highway, North Vancouver, British Columbia V7H 1V2 (Canada); Osachoff, Heather [Pacific Environmental Science Centre, 2645 Dollarton Highway, North Vancouver, British Columbia V7H 1V2 (Canada); Wigmore, Heidi [Pacific Environmental Science Centre, 2645 Dollarton Highway, North Vancouver, British Columbia V7H 1V2 (Canada); Clapson, David J. [Department of Biochemistry and Microbiology, P.O. Box 3055, Stn. CSC, University of Victoria, Victoria, British Columbia V8W 3P6 (Canada); Gunderson, Mark P. [Department of Biochemistry and Microbiology, P.O. Box 3055, Stn. CSC, University of Victoria, Victoria, British Columbia V8W 3P6 (Canada); Van Aggelen, Graham [Pacific Environmental Science Centre, 2645 Dollarton Highway, North Vancouver, British Columbia V7H 1V2 (Canada); Helbing, Caren C. [Department of Biochemistry and Microbiology, P.O. Box 3055, Stn. CSC, University of Victoria, Victoria, British Columbia V8W 3P6 (Canada)]. E-mail: chelbing@uvic.ca

    2006-12-01

    We investigated whether exposure to environmentally relevant concentrations of the bactericidal agent, triclosan, induces changes in the thyroid hormone-mediated process of metamorphosis of the North American bullfrog, Rana catesbeiana and alters the expression profile of thyroid hormone receptor (TR) {alpha} and {beta}, basic transcription element binding protein (BTEB) and proliferating nuclear cell antigen (PCNA) gene transcripts. Premetamorphic tadpoles were immersed in environmentally relevant concentrations of triclosan and injected with 1 x 10{sup -11} mol/g body weight 3,5,3'-triiodothyronine (T{sub 3}) or vehicle control. Morphometric measurements and steady-state mRNA levels obtained by quantitative polymerase chain reaction were determined. mRNA abundance was also examined in Xenopus laevis XTC-2 cells treated with triclosan and/or 10 nM T{sub 3}. Tadpoles pretreated with triclosan concentrations as low as 0.15 {+-} 0.03 {mu}g/L for 4 days showed increased hindlimb development and a decrease in total body weight following T{sub 3} administration. Triclosan exposure also resulted in decreased T{sub 3}-mediated TR{beta} mRNA expression in the tadpole tail fin and increased levels of PCNA transcript in the brain within 48 h of T{sub 3} treatment whereas TR{alpha} and BTEB were unaffected. Triclosan alone altered thyroid hormone receptor {alpha} transcript levels in the brain of premetamorphic tadpoles and induced a transient weight loss. In XTC-2 cells, exposure to T{sub 3} plus nominal concentrations of triclosan as low as 0.03 {mu}g/L for 24 h resulted in altered thyroid hormone receptor mRNA expression. Exposure to low levels of triclosan disrupts thyroid hormone-associated gene expression and can alter the rate of thyroid hormone-mediated postembryonic anuran development.

  4. Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes.

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    Ben C L van Schaijk

    Full Text Available Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.

  5. Conversion of homothallic yeast to heterothallism trough HO gene disruption

    CSIR Research Space (South Africa)

    Van Zyl, WH

    1993-04-01

    Full Text Available A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeaststrains to heterothallism through HO gene disruption. An integrative ho:: neo disrupted allele was constructed by cloning a dominant selectable marker...

  6. Conversion of homothallic yeast to heterothallism through to gene disruption

    CSIR Research Space (South Africa)

    Van Zyl, WH

    1993-04-01

    Full Text Available A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho=neo disrupted allele was constructed by cloning a dominant selectable marker...

  7. Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases.

    Science.gov (United States)

    Preciados, Mark; Yoo, Changwon; Roy, Deodutta

    2016-12-13

    During the development of an individual from a single cell to prenatal stages to adolescence to adulthood and through the complete life span, humans are exposed to countless environmental and stochastic factors, including estrogenic endocrine disrupting chemicals. Brain cells and neural circuits are likely to be influenced by estrogenic endocrine disruptors (EEDs) because they strongly dependent on estrogens. In this review, we discuss both environmental, epidemiological, and experimental evidence on brain health with exposure to oral contraceptives, hormonal therapy, and EEDs such as bisphenol-A (BPA), polychlorinated biphenyls (PCBs), phthalates, and metalloestrogens, such as, arsenic, cadmium, and manganese. Also we discuss the brain health effects associated from exposure to EEDs including the promotion of neurodegeneration, protection against neurodegeneration, and involvement in various neurological deficits; changes in rearing behavior, locomotion, anxiety, learning difficulties, memory issues, and neuronal abnormalities. The effects of EEDs on the brain are varied during the entire life span and far-reaching with many different mechanisms. To understand endocrine disrupting chemicals mechanisms, we use bioinformatics, molecular, and epidemiologic approaches. Through those approaches, we learn how the effects of EEDs on the brain go beyond known mechanism to disrupt the circulatory and neural estrogen function and estrogen-mediated signaling. Effects on EEDs-modified estrogen and nuclear respiratory factor 1 (NRF1) signaling genes with exposure to natural estrogen, pharmacological estrogen-ethinyl estradiol, PCBs, phthalates, BPA, and metalloestrogens are presented here. Bioinformatics analysis of gene-EEDs interactions and brain disease associations identified hundreds of genes that were altered by exposure to estrogen, phthalate, PCBs, BPA or metalloestrogens. Many genes modified by EEDs are common targets of both 17 β-estradiol (E2) and NRF1. Some of

  8. Estrogenic Endocrine Disrupting Chemicals Influencing NRF1 Regulated Gene Networks in the Development of Complex Human Brain Diseases

    Directory of Open Access Journals (Sweden)

    Mark Preciados

    2016-12-01

    Full Text Available During the development of an individual from a single cell to prenatal stages to adolescence to adulthood and through the complete life span, humans are exposed to countless environmental and stochastic factors, including estrogenic endocrine disrupting chemicals. Brain cells and neural circuits are likely to be influenced by estrogenic endocrine disruptors (EEDs because they strongly dependent on estrogens. In this review, we discuss both environmental, epidemiological, and experimental evidence on brain health with exposure to oral contraceptives, hormonal therapy, and EEDs such as bisphenol-A (BPA, polychlorinated biphenyls (PCBs, phthalates, and metalloestrogens, such as, arsenic, cadmium, and manganese. Also we discuss the brain health effects associated from exposure to EEDs including the promotion of neurodegeneration, protection against neurodegeneration, and involvement in various neurological deficits; changes in rearing behavior, locomotion, anxiety, learning difficulties, memory issues, and neuronal abnormalities. The effects of EEDs on the brain are varied during the entire life span and far-reaching with many different mechanisms. To understand endocrine disrupting chemicals mechanisms, we use bioinformatics, molecular, and epidemiologic approaches. Through those approaches, we learn how the effects of EEDs on the brain go beyond known mechanism to disrupt the circulatory and neural estrogen function and estrogen-mediated signaling. Effects on EEDs-modified estrogen and nuclear respiratory factor 1 (NRF1 signaling genes with exposure to natural estrogen, pharmacological estrogen-ethinyl estradiol, PCBs, phthalates, BPA, and metalloestrogens are presented here. Bioinformatics analysis of gene-EEDs interactions and brain disease associations identified hundreds of genes that were altered by exposure to estrogen, phthalate, PCBs, BPA or metalloestrogens. Many genes modified by EEDs are common targets of both 17 β-estradiol (E2 and

  9. Disruption of Four Kinesin Genes in Dictyostelium

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    Soga Ikko

    2008-04-01

    Full Text Available Abstract Background Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans. Results We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family and Kif10 (kinesin-8; Kip3 family appear to cooperate with dynein to organize the interphase radial microtubule array. Conclusion The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family.

  10. Gene disruption using zinc finger nuclease technology.

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    Granja, Sara; Marchiq, Ibtissam; Baltazar, Fátima; Pouysségur, Jacques

    2014-01-01

    Zinc finger nucleases are reagents that induce DNA double-strand breaks at specific sites that can be repaired by nonhomologous end joining, inducing alterations in the genome. This strategy has enabled highly efficient gene disruption in numerous cell types and model organisms opening a door for new therapeutic applications. Here, we describe the disruption of CD147/basigin by this technique in a human cancer cell line.

  11. Silencing abnormal wing disc gene of the Asian citrus psyllid, Diaphorina citri disrupts adult wing development and increases nymph mortality.

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    Ibrahim El-Shesheny

    Full Text Available Huanglongbing (HLB causes considerable economic losses to citrus industries worldwide. Its management depends on controlling of the Asian citrus Psyllid (ACP, the vector of the bacterium, Candidatus Liberibacter asiaticus (CLas, the causal agent of HLB. Silencing genes by RNA interference (RNAi is a promising tool to explore gene functions as well as control pests. In the current study, abnormal wing disc (awd gene associated with wing development in insects is used to interfere with the flight of psyllids. Our study showed that transcription of awd is development-dependent and the highest level was found in the last instar (5(th of the nymphal stage. Micro-application (topical application of dsRNA to 5(th instar of nymphs caused significant nymphal mortality and adult wing-malformation. These adverse effects in ACP were positively correlated with the amounts of dsRNA used. A qRT-PCR analysis confirmed the dsRNA-mediated transcriptional down-regulation of the awd gene. Significant down-regulation was required to induce a wing-malformed phenotype. No effect was found when dsRNA-gfp was used, indicating the specific effect of dsRNA-awd. Our findings suggest a role for awd in ACP wing development and metamorphosis. awd could serve as a potential target for insect management either via direct application of dsRNA or by producing transgenic plants expressing dsRNA-awd. These strategies will help to mitigate HLB by controlling ACP.

  12. Disruption of neurogenesis and cortical development in transgenic mice misexpressing Olig2, a gene in the Down syndrome critical region.

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    Liu, Wei; Zhou, Hui; Liu, Lei; Zhao, Chuntao; Deng, Yaqi; Chen, Lina; Wu, Laiman; Mandrycky, Nicole; McNabb, Christopher T; Peng, Yuanbo; Fuchs, Perry N; Lu, Jie; Sheen, Volney; Qiu, Mengsheng; Mao, Meng; Lu, Q Richard

    2015-05-01

    The basic helix-loop-helix (bHLH) transcription factor Olig2 is crucial for mammalian central nervous system development. Human ortholog OLIG2 is located in the Down syndrome critical region in trisomy 21. To investigate the effect of Olig2 misexpression on brain development, we generated a developmentally regulated Olig2-overexpressing transgenic line with a Cre/loxP system. The transgenic mice with Olig2 misexpression in cortical neural stem/progenitor cells exhibited microcephaly, cortical dyslamination, hippocampus malformation, and profound motor deficits. Ectopic misexpression of Olig2 impaired cortical progenitor proliferation and caused precocious cell cycle exit. Massive neuronal cell death was detected in the developing cortex of Olig2-misexpressing mice. In addition, Olig2 misexpression led to a significant downregulation of neuronal specification factors including Ngn1, Ngn2 and Pax6, and a defect in cortical neurogenesis. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis indicates that Olig2 directly targets the promoter and/or enhancer regions of Nfatc4, Dscr1/Rcan1 and Dyrk1a, the critical neurogenic genes that contribute to Down syndrome phenotypes, and inhibits their expression. Together, our study suggests that Olig2 misexpression in neural stem cells elicits neurogenesis defects and neuronal cell death, which may contribute to developmental disorders including Down syndrome, where OLIG2 is triplicated on chromosomal 21. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development.

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    Piya, Anil; Kaur, Jasmeet; Rice, Alan M; Bose, Himangshu S

    2017-01-01

    Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR), which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH). Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon-intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo. STAR exon 1 deletion caused lipoid CAH.Exon 1 substitution does not affect biochemical activity.StAR promoter is responsible for gonadal development.

  14. De novo disruption of promoter and exon 1 of STAR gene reveals essential role for gonadal development

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    Anil Piya

    2017-03-01

    Full Text Available Cholesterol transport into the mitochondria is required for synthesis of the first steroid, pregnenolone. Cholesterol is transported by the steroidogenic acute regulatory protein (STAR, which acts at the outer mitochondrial membrane prior to its import. Mutations in the STAR protein result in lipoid congenital adrenal hyperplasia (CAH. Although the STAR protein consists of seven exons, biochemical analysis in nonsteroidogenic COS-1 cells showed that the first two were not essential for pregnenolone synthesis. Here, we present a patient with ambiguous genitalia, salt-lossing crisis within two weeks after birth and low cortisol levels. Sequence analysis of the STAR, including the exon–intron boundaries, showed the complete deletion of exon 1 as well as more than 50 nucleotides upstream of STAR promoter. Mitochondrial protein import with the translated protein through synthesis cassette of the mutant STAR lacking exon 1 showed protein translation, but it is less likely to have synthesized without a promoter in our patient. Thus, a full-length STAR gene is necessary for physiological mitochondrial cholesterol transport in vivo.

  15. Overexpression and Knockdown of Hypoxia-Inducible Factor 1 Disrupt the Expression of Steroidogenic Enzyme Genes and Early Embryonic Development in Zebrafish

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    Tianfeng Tan

    2017-06-01

    Full Text Available Hypoxia is an important environmental stressor leading to endocrine disruption and reproductive impairment in fish. Although the hypoxia-inducible factor 1 (HIF-1 is known to regulate the transcription of various genes mediating oxygen homeostasis, its role in modulating steroidogenesis-related gene expression remains poorly understood. In this study, the regulatory effect of HIF-1 on the expression of 9 steroidogenic enzyme genes was investigated in zebrafish embryos using a “gain-of-function and loss-of-function” approach. Eight of the genes, CYP11a, CYP11b2, 3β-HSD, HMGCR, CYP17a1, 17β-HSD2, CYP19a , and CYP19b , were found to be differentially upregulated at 24 and 48 hpf following zHIF-1α-ΔODD overexpression (a mutant zebrafish HIF-1α protein with proline-414 and proline-557 deleted. Knockdown of zHIF-1α also affected the expression pattern of the steroidogenic enzyme genes. Overexpression of zHIF-1α and hypoxia exposure resulted in downregulated StAR expression but upregulated CYP11a and 3β-HSD expression in zebrafish embryos. Conversely, the expression patterns of these 3 genes were reversed in embryos in which zHIF-1α was knocked down under normoxia, suggesting that these 3 genes are regulated by HIF-1. Overall, the findings from this study indicate that HIF-1–mediated mechanisms are likely involved in the regulation of specific steroidogenic genes.

  16. Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity

    NARCIS (Netherlands)

    Ra, Runar; Beerthuyzen, Marke M.; Vos, Willem M. de; Saris, Per E.J.; Kuipers, Oscar P.

    1999-01-01

    The lantibiotic nisin is produced by several strains of Lactococcus lactis subsp. lactis. The chromosomally located gene cluster nisABTCIPRKFEG is required for biosynthesis, development of immunity, and regulation of gene expression. In-frame deletions in the nisB and nisT genes, and disruption of

  17. Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

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    Pateau, Victoire; Razafimandimby, Bienvenue; Vandeputte, Patrick; Thornton, Christopher R; Guillemette, Thomas; Bouchara, Jean-Philippe; Giraud, Sandrine

    2017-10-11

    Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

  18. RNA interference can be used to disrupt gene function in tardigrades.

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    Tenlen, Jennifer R; McCaskill, Shaina; Goldstein, Bob

    2013-05-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.

  19. Symbiotic Gene Activation is Interrupted by Endocrine Disrupting Chemicals

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    Jennifer E. Fox

    2001-01-01

    Full Text Available Endocrine disrupting chemicals (EDCs include organochlorine pesticides, plastics manufacturing by-products, and certain herbicides[1]. These chemicals have been shown to disrupt hormonal signaling in exposed wildlife, lab animals, and mammalian cell culture by binding to estrogen receptors (ER-α and ER-β and affecting the expression of estrogen responsive genes[2,3]. Additionally, certain plant chemicals, termed phytoestrogens, are also able to bind to estrogen receptors and modulate gene expression, and as such also may be considered EDCs[4]. One example of phytoestrogen action is genistein, a phytochemical produced by soybeans, binding estrogen receptors, and changing expression of estrogen responsive genes which certain studies have linked to a lower incidence of hormonally related cancers in Japanese populations[5]. Why would plants make compounds that are able to act as estrogens in the human body? Obviously, soybeans do not intentionally produce phytoestrogens to prevent breast cancer in Japanese women.

  20. The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

    Energy Technology Data Exchange (ETDEWEB)

    Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P. Robin; Schulze, Karen L.; Rubin, Gerald M.; Hoskins, Roger A.; Spradling, Allan C.

    2004-01-13

    The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

  1. Bisphenol A, an endocrine-disrupting chemical, and brain development.

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    Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2012-08-01

    Bisphenol A (BPA) is an endocrine-disrupting chemical, widely used in various industries and the field of dentistry. The consequent increase in BPA exposure among humans has led us to some concerns regarding the potential deleterious effects on reproduction and brain development. The emphasis of this review is on the effects of prenatal and lactational exposure to low doses of BPA on brain development in mice. We demonstrated that prenatal exposure to BPA affected fetal murine neocortical development by accelerating neuronal differentiation/migration during the early embryonic stage, which was associated with up- and down-regulation of the genes critical for brain development, including the basic helix-loop-helix transcription factors. In the adult mice brains, both abnormal neocortical architecture and abnormal corticothalamic projections persisted in the group exposed to the BPA. Functionally, BPA exposure disturbed murine behavior, accompanied with a disrupted neurotransmitter system, including monoamines, in the postnatal development period and in adult mice. We also demonstrated that epigenetic alterations in promoter-associated CpG islands might underlie some of the effects on brain development after exposure to BPA. © 2012 Japanese Society of Neuropathology.

  2. Disruption of zebrafish cyclin G-associated kinase (GAK function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development

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    Szeto Daniel P

    2010-01-01

    Full Text Available Abstract Background The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish. Results Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK, differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures. Conclusion In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

  3. PARK2, a Large Common Fragile Site Gene, is Part of a Stress Response Network in Normal Cells that is Disrupted During the Development of Ovarian Cancer

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    2008-01-01

    Zimonjic DB, Druck T, Ohta M, Kastury K, Croce CM, Popescu NC, Huebner K. Positions of chromosome 3p14.2 fragile sites (FRA3B) within the FHIT gene. Cancer...common fragile site gene, is involved in cellular stress response. Oncogene2006; 25: 2901-2908. 15) Sirashi T, Druck T, Mimori K, Flomenberg J

  4. Gene-specific disruption of endocannabinoid receptor 1 (cnr1a) by ethanol probably leads to the development of fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish (Oryzias latipes) embryogenesis.

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    Dasmahapatra, Asok K; Khan, Ikhlas A

    2015-01-01

    The present study was designed to investigate the probable roles played by cannabinoid (CB) receptors in fetal alcohol spectrum disorder (FASD) induction in Japanese rice fish (Oryzias latipes). Searching of public databases (GenBank, Ensembl) indicated that the Japanese rice fish genome includes three human ortholog CB receptor genes (cnr1a, cnr1b and cnr2). Quantitative real-time PCR (qPCR) and whole mount in situ hybridization (WMISH) techniques were used to analyze the expression of these cnr genes during Japanese rice fish embryogenesis and also in response to developmental ethanol exposure. qPCR analyses showed that the expression of all three CB receptor genes were developmentally regulated and only cnr2 showed maternal expression. The mRNA concentrations of these genes were found to be enhanced after 3 dpf and attained maximal levels either prior to or after hatching. WMISH technique indicated that all three cnr genes were expressed in the head region of hatchlings. During development, ethanol selectively attenuated the expression of cnr1a mRNA only. Blocking of cnr1a mRNA by CB1 receptor antagonists rimonabant (10-20 μM) or AM251 (0.2-1 μM) 0-2 dpf were unable to induce any FASD-related phenotypic features in embryos or in hatchlings. However, continuous exposure of the embryos (0-6 dpf) to AM251 (1 μM) was able to reduce the hatching efficiency of the embryos. Our data indicated that in Japanese rice fish, ethanol disrupted the expression of only cnr1a in a concentration-dependent manner that induced delay in hatching and might be responsible for the development of FASD phenotypes. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. USP40 gene knockdown disrupts glomerular permeability in zebrafish.

    Science.gov (United States)

    Takagi, Hisashi; Nishibori, Yukino; Katayama, Kan; Katada, Tomohisa; Takahashi, Shohei; Kiuchi, Zentaro; Takahashi, Shin-Ichiro; Kamei, Hiroyasu; Kawakami, Hayato; Akimoto, Yoshihiro; Kudo, Akihiko; Asanuma, Katsuhiko; Takematsu, Hiromu; Yan, Kunimasa

    2017-04-01

    Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis. Copyright © 2017 the American Physiological Society.

  6. Disrupted tRNA Genes and tRNA Fragments: A Perspective on tRNA Gene Evolution

    Directory of Open Access Journals (Sweden)

    Akio Kanai

    2015-01-01

    Full Text Available Transfer RNAs (tRNAs are small non-coding RNAs with lengths of approximately 70–100 nt. They are directly involved in protein synthesis by carrying amino acids to the ribosome. In this sense, tRNAs are key molecules that connect the RNA world and the protein world. Thus, study of the evolution of tRNA molecules may reveal the processes that led to the establishment of the central dogma: genetic information flows from DNA to RNA to protein. Thanks to the development of DNA sequencers in this century, we have determined a huge number of nucleotide sequences from complete genomes as well as from transcriptomes in many species. Recent analyses of these large data sets have shown that particular tRNA genes, especially in Archaea, are disrupted in unique ways: some tRNA genes contain multiple introns and some are split genes. Even tRNA molecules themselves are fragmented post-transcriptionally in many species. These fragmented small RNAs are known as tRNA-derived fragments (tRFs. In this review, I summarize the progress of research into the disrupted tRNA genes and the tRFs, and propose a possible model for the molecular evolution of tRNAs based on the concept of the combination of fragmented tRNA halves.

  7. Disruption of the GH Receptor Gene in Adult Mice Increases Maximal Lifespan in Females

    DEFF Research Database (Denmark)

    Junnila, Riia K.; Duran-Ortiz, Silvana; Suer, Ozan

    2016-01-01

    GH and IGF-1 are important for a variety of physiological processes including growth, development, and aging. Mice with reduced levels of GH and IGF-1 have been shown to live longer than wild-type controls. Our laboratory has previously found that mice with a GH receptor gene knockout (GHRKO) from...... affect metabolism and longevity. Thus, we produced adult-onset GHRKO (aGHRKO) mice by disrupting the Ghr gene at 6 weeks of age. We found that aGHRKO mice replicate many of the beneficial effects observed in long-lived GHRKO mice. For example, aGHRKO mice, like GHRKO animals, displayed retarded growth...... carry germline mutations. Importantly, the effect of a long-term suppression of the GH/IGF-1 axis during adulthood, as would be considered for human therapeutic purposes, has not been tested. The goal of this study was to determine whether temporally controlled Ghr gene deletion in adult mice would...

  8. Low load for disruptive mutations in autism genes and their biased transmission

    Science.gov (United States)

    Iossifov, Ivan; Levy, Dan; Allen, Jeremy; Ye, Kenny; Ronemus, Michael; Lee, Yoon-ha; Yamrom, Boris; Wigler, Michael

    2015-01-01

    We previously computed that genes with de novo (DN) likely gene-disruptive (LGD) mutations in children with autism spectrum disorders (ASD) have high vulnerability: disruptive mutations in many of these genes, the vulnerable autism genes, will have a high likelihood of resulting in ASD. Because individuals with ASD have lower fecundity, such mutations in autism genes would be under strong negative selection pressure. An immediate prediction is that these genes will have a lower LGD load than typical genes in the human gene pool. We confirm this hypothesis in an explicit test by measuring the load of disruptive mutations in whole-exome sequence databases from two cohorts. We use information about mutational load to show that lower and higher intelligence quotients (IQ) affected individuals can be distinguished by the mutational load in their respective gene targets, as well as to help prioritize gene targets by their likelihood of being autism genes. Moreover, we demonstrate that transmission of rare disruptions in genes with a lower LGD load occurs more often to affected offspring; we show transmission originates most often from the mother, and transmission of such variants is seen more often in offspring with lower IQ. A surprising proportion of transmission of these rare events comes from genes expressed in the embryonic brain that show sharply reduced expression shortly after birth. PMID:26401017

  9. Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

    Science.gov (United States)

    Taylor, Cathy J; McRobert, Louisa; Baker, David A

    2008-01-01

    Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation – a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation – the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCβ gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEδ gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEδ plays a crucial role in regulating cGMP levels during sexual development. PMID:18452584

  10. New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe.

    Science.gov (United States)

    Sato, Masamitsu; Dhut, Susheela; Toda, Takashi

    2005-05-01

    We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (Bähler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of protein functions in fission yeast.

  11. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  12. Do Thyroid Disrupting Chemicals Influence Foetal Development during Pregnancy?

    DEFF Research Database (Denmark)

    Hartoft-Nielsen, Marie-Louise; Boas, Malene; Bliddal, Sofie

    2011-01-01

    Maternal euthyroidism during pregnancy is crucial for normal development and, in particular, neurodevelopment of the foetus. Up to 3.5 percent of pregnant women suffer from hypothyroidism. Industrial use of various chemicals-endocrine disrupting chemicals (EDCs)-has been shown to cause almost...

  13. CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

    Science.gov (United States)

    Kalebic, Nereo; Taverna, Elena; Tavano, Stefania; Wong, Fong Kuan; Suchold, Dana; Winkler, Sylke; Huttner, Wieland B; Sarov, Mihail

    2016-03-01

    We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈ 90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  14. An Epigenetic Role for Disrupted Paternal Gene Expression in Postzygotic Seed Abortion in Arabidopsis Interspecific Hybrids.

    Science.gov (United States)

    Kirkbride, Ryan C; Yu, Helen Hong; Nah, Gyoungju; Zhang, Changqing; Shi, Xiaoli; Chen, Z Jeffrey

    2015-12-07

    Interspecific hybrids often increase the levels of heterozygosity and hybrid vigor, but some interspecific hybrid seeds are aborted shortly after fertilization. The mechanism behind this postzygotic seed abortion is poorly understood. Here, we report genome-wide analysis of allelic expression changes in developing siliques and seeds in three F1 interspecific crosses between Arabidopsis thaliana (Col, Ler, or C24) and Arabidopsis arenosa. The majority of maternally expressed genes (MEGs) were shared among all three F1 interspecific crosses, whereas ∼90% of 272 paternally expressed genes (PEGs) were found only in one or two F1 crosses, suggesting a role for disrupted paternal gene expression in seed abortion that varies in different crosses. Consistent with this notion, 12 PEGs in the infertile interspecific hybrids matched MEGs in fertile intraspecific hybrids. This disruption of PEGs in the interspecific hybrids was consistent with the upregulation of the genes in the paternal-excess interploidy cross (2X6) between a diploid mother and a hexaploid father, leading to the seed abortion. Moreover, a subset of PEGs in the interspecific crosses were also upregulated in the intraspecific hybrid met1XWT or meaXWT, in which the mutant of MET1 (DNA METHYLTRANSFERASE1) or MEDEA, a Polycomb Repressive Complex2 gene, was used as the maternal parent. These data suggest that maternal epigenetic factors and paternal gene expression play important roles in the postzygotic seed abortion in interspecific hybrids or neo-allopolyploids. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  15. Conversion of the pathogenic fungus Colletotrichum magna to a nonpathogenic, endophytic mutualist by gene disruption

    Science.gov (United States)

    Redman, R.S.; Ranson, J.C.; Rodriguez, R.J.

    1999-01-01

    Hygromycin-resistant transformants of the cucurbit pathogen Colletotrichum magna (teleomorph: Glomerella magna) were generated by restriction enzyme-mediated integration (REMI) transformation. A rapid pathogenicity assay involving watermelon (Citrullus lanatus) seedlings was developed and 14,400 REMI transformants were screened and assessed for their ability to cause disease, colonize plant tissues, and confer disease resistance against wild-type C. magna. A total of 176 nonpathogenic REMI mutants capable of colonizing cucurbit plants were isolated and assigned to three groups based on their ability to confer disease resistance: phenotype A, 80 to 100% disease protection; phenotype B, 10 to 65% disease protection; and phenotype C, 0 to 4% disease protection. Molecular and genetic analyses of one REMI mutant (R1) indicated that the nonpathogenic phenotype A resulted from a single-site integration. R1 showed a 1:1 segregation of hygromycin resistance and nonpathogenicity and all hygromycin-resistant progeny were nonpathogenic. The integrated vector and 5.5 kb of flanking fungal genomic DNA were isolated from R1 and designated pGMR1. To verify that pGMR1 contained pathogenicity gene sequences, a wild-type isolate of C. magna was transformed with pGMR1 to induce gene disruptions by homologous integration. Approximately 47% of the pGMR1 transformants expressed phenotype A, indicating homologous integration and gene disruption.

  16. [Sleep/wake cycle, circadian disruption and the development of obesity].

    Science.gov (United States)

    Masaki, Takayuki

    2012-07-01

    It is increasingly recognized that obesity is an important health problem. The mechanisms that underlie obesity have not been fully elucidated, and effective therapeutic approaches are currently of general interest. Recent studies have provided evidence that circadian clock is a crucial factor in the development of obesity and related metabolic disease. Genetic disruption of clock genes in mice displayed metabolic dysfunctions of specific tissues at distinct phases of the sleep/wake cycle. In addition, circadian desynchrony, a characteristic of shift work and short sleep, are associated with obesity in human. Here, I describe the advances in understanding the interrelationship among circadian disruption, sleep deprivation and obesity.

  17. Disrupted postnatal lung development in heme oxygenase-1 deficient mice

    Directory of Open Access Journals (Sweden)

    Zhang Huayan

    2010-10-01

    Full Text Available Abstract Background Heme oxygenase (HO degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development. Methods Neonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex till postnatal 14 days, and evaluated for lung development. Results Compared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment. Conclusions These experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development.

  18. Disruption of clock gene expression in human colorectal liver metastases

    NARCIS (Netherlands)

    S.A. Huisman (Sander); K.R. Ahmadi (Kourosh); J.N.M. IJzermans (Jan); C. Verhoef (Kees); G.T.J. van der Horst (Gijsbertus); R.W.F. de Bruin (Ron)

    2016-01-01

    textabstractThe circadian timing system controls about 40 % of the transcriptome and is important in the regulation of a wide variety of biological processes including metabolic and proliferative functions. Disruption of the circadian clock could have significant effect on human health and has an

  19. Disruption of immunological tolerance: role of AIRE gene in autoimmunity.

    Science.gov (United States)

    Rizzi, M; Ferrera, F; Filaci, G; Indiveri, F

    2006-02-01

    The mechanism underlying the generation of T and B autoreactive clones in autoimmune diseases is still unknown. Among genetic factors implicated in autoimmunity, Autoimmune Regulator gene (AIRE) is one of the candidates to better understand the complex scenario of autoimmune manifestations. AIRE mutations are responsible for the development of autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) with monogenic autosomal recessive inheritance; it has been shown that AIRE regulates the negative selection of autoreactive T cells clones, driving the transcription of tissue-specific antigens in thymic epithelial cells. In various autoimmune manifestations correlated or not to APECED, AIRE variants act in a semidominant manner, leading to a reduction in AIRE protein amount per cell, and consequently to a marked decrease in ectopic proteins expression in the thymus. The co-occurrence of autoimmune diseases in the same individual has prompted several studies aimed to recognize shared patho-physiological mechanisms; in this scenario small reductions in function could explain the predisposition to autoimmunity in AIRE-heterozygous carriers of missense mutations; further studies to investigate whether the AIRE gene is involved in determining these autoimmune manifestations should be carried out.

  20. Maternal obesity disrupts circadian rhythms of clock and metabolic genes in the offspring heart and liver.

    Science.gov (United States)

    Wang, Danfeng; Chen, Siyu; Liu, Mei; Liu, Chang

    2015-06-01

    Early life nutritional adversity is tightly associated with the development of long-term metabolic disorders. Particularly, maternal obesity and high-fat diets cause high risk of obesity in the offspring. Those offspring are also prone to develop hyperinsulinemia, hepatic steatosis and cardiovascular diseases. However, the precise underlying mechanisms leading to these metabolic dysregulation in the offspring remain unclear. On the other hand, disruptions of diurnal circadian rhythms are known to impair metabolic homeostasis in various tissues including the heart and liver. Therefore, we investigated that whether maternal obesity perturbs the circadian expression rhythms of clock, metabolic and inflammatory genes in offspring heart and liver by using RT-qPCR and Western blotting analysis. Offspring from lean and obese dams were examined on postnatal day 17 and 35, when pups were nursed by their mothers or took food independently. On P17, genes examined in the heart either showed anti-phase oscillations (Cpt1b, Pparα, Per2) or had greater oscillation amplitudes (Bmal1, Tnf-α, Il-6). Such phase abnormalities of these genes were improved on P35, while defects in amplitudes still existed. In the liver of 17-day-old pups exposed to maternal obesity, the oscillation amplitudes of most rhythmic genes examined (except Bmal1) were strongly suppressed. On P35, the oscillations of circadian and inflammatory genes became more robust in the liver, while metabolic genes were still kept non-rhythmic. Maternal obesity also had a profound influence in the protein expression levels of examined genes in offspring heart and liver. Our observations indicate that the circadian clock undergoes nutritional programing, which may contribute to the alternations in energy metabolism associated with the development of metabolic disorders in early life and adulthood.

  1. Do Thyroid Disrupting Chemicals Influence Foetal Development during Pregnancy?

    DEFF Research Database (Denmark)

    Hartoft-Nielsen, Marie-Louise; Boas, Malene; Bliddal, Sofie

    2011-01-01

    Maternal euthyroidism during pregnancy is crucial for normal development and, in particular, neurodevelopment of the foetus. Up to 3.5 percent of pregnant women suffer from hypothyroidism. Industrial use of various chemicals-endocrine disrupting chemicals (EDCs)-has been shown to cause almost...... constant exposure of humans with possible harmful influence on health and hormone regulation. EDCs may affect thyroid hormone homeostasis by different mechanisms, and though the effect of each chemical seems scarce, the added effects may cause inappropriate consequences on, for example, foetal...... neurodevelopment. This paper focuses on thyroid hormone influence on foetal development in relation to the chemicals suspected of thyroid disrupting properties with possible interactions with maternal thyroid homeostasis. Knowledge of the effects is expected to impact the general debate on the use...

  2. Do Thyroid Disrupting Chemicals Influence Foetal Development during Pregnancy?

    Directory of Open Access Journals (Sweden)

    Marie-Louise Hartoft-Nielsen

    2011-01-01

    Full Text Available Maternal euthyroidism during pregnancy is crucial for normal development and, in particular, neurodevelopment of the foetus. Up to 3.5 percent of pregnant women suffer from hypothyroidism. Industrial use of various chemicals—endocrine disrupting chemicals (EDCs—has been shown to cause almost constant exposure of humans with possible harmful influence on health and hormone regulation. EDCs may affect thyroid hormone homeostasis by different mechanisms, and though the effect of each chemical seems scarce, the added effects may cause inappropriate consequences on, for example, foetal neurodevelopment. This paper focuses on thyroid hormone influence on foetal development in relation to the chemicals suspected of thyroid disrupting properties with possible interactions with maternal thyroid homeostasis. Knowledge of the effects is expected to impact the general debate on the use of these chemicals. However, more studies are needed to elucidate the issue, since human studies are scarce.

  3. Disruption of the neurexin 1 gene is associated with schizophrenia

    DEFF Research Database (Denmark)

    Rujescu, Dan; Ingason, Andres; Cichon, Sven

    2009-01-01

    Deletions within the neurexin 1 gene (NRXN1; 2p16.3) are associated with autism and have also been reported in two families with schizophrenia. We examined NRXN1, and the closely related NRXN2 and NRXN3 genes, for copy number variants (CNVs) in 2977 schizophrenia patients and 33 746 controls from...

  4. Disruptiveness, Friends' Characteristics, and Delinquency in Early Adolescence: A Test of Two Competing Models of Development.

    Science.gov (United States)

    Vitaro, Frank; Tremblay, Richard E.; Kerr, Margaret; Pagani, Linda; Bukowski, William M.

    1997-01-01

    Tested the individual characteristics and deviant peer association theoretical models of friends' influence on the development of delinquency in disruptive boys. Found that moderately disruptive boys with aggressive-disturbing friends were more delinquent at age 13 than other subgroups of moderately disruptive boys. Highly disruptive and…

  5. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos

    Directory of Open Access Journals (Sweden)

    Ken-ichi T. Suzuki

    2013-03-01

    Recently, gene editing with transcription activator-like effector nucleases (TALENs has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.

  6. Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

    Science.gov (United States)

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

  7. Playware ABC 2: a Disruptive Technology for Global Development

    DEFF Research Database (Denmark)

    Lund, Henrik Hautop

    2017-01-01

    The Playware ABC concept is used to create solutions that are usable by all kinds of users and contexts in our globalized society. In this paper, the Playware ABC can be exemplified with the development of the modular interactive tiles for health prevention and rehabilitation of anybody, anywhere......, anytime. The paper gives examples of how playware becomes a disruptive technology for global development, for instance in the health sector. For instance, in Tanzania doctors and community-based rehabilitation workers are constructing and combining modular playware tiles to easily create the right kind...... of intervention for their patients in both urban and deep rural areas in Tanzania....

  8. Disrupting the circadian clock: gene-specific effects on aging, cancer, and other phenotypes.

    Science.gov (United States)

    Yu, Elizabeth A; Weaver, David R

    2011-05-01

    The circadian clock imparts 24-hour rhythmicity on gene expression and cellular physiology in virtually all cells. Disruption of the genes necessary for the circadian clock to function has diverse effects, including aging-related phenotypes. Some circadian clock genes have been described as tumor suppressors, while other genes have less clear functions in aging and cancer. In this Review, we highlight a recent study [Dubrovsky et al., Aging 2: 936-944, 2010] and discuss the much larger field examining the relationship between circadian clock genes, circadian rhythmicity, aging-related phenotypes, and cancer.

  9. Endocrine disrupting compounds exposure and testis development in mammals

    Science.gov (United States)

    Egbowona, Biola F.; Mustapha, Olajide A.

    2011-01-01

    In the last few decades, there is substantial evidence that male reproductive function is deteriorating in humans and wildlife and this is associated with unintentional exposure to widely used synthetic chemicals. Subsequently, much has been done to show that certain chemicals in the environment adversely interfere with the developing fetal gonads of the laboratory animals. Some in vitro studies have demonstrated treatment-induced reproductive problems in offspring exposed to endocrine disrupting compounds (EDC) which are similar to those observed in wildlife and human population. Few EDC studies have demonstrated that there are certain periods of gestation when the developing fetus is highly sensitive and at risk of small endocrine changes. Similar observations have been made in the sewage sludge model, however, while animal studies have been insightful in providing valuable information about the range of effects that can be attributed to in utero exposure to EDCs, varying levels of maternal doses administered in different studies exaggerated extrapolation of these results to human. Thus the EDC concentration representative of fetal exposure levels is uncertain because of the complexities of its nature. So far, the level of fetal exposure can only be roughly estimated. There is substantial evidence from animal data to prove that EDCs can adversely affect reproductive development and function in male and more has accumulated on the mechanisms by which they exert their effects. This paper therefore, reviews previous studies to highlight the extent to which testis development can be disrupted during fetal life. PMID:29255381

  10. Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription.

    Science.gov (United States)

    Žumer, Kristina; Plemenitaš, Ana; Saksela, Kalle; Peterlin, B Matija

    2011-10-01

    Autoimmune regulator (AIRE) is a transcription factor that induces the expression of a large subset of otherwise strictly tissue restricted antigens in medullary thymic epithelial cells, thereby enabling their presentation to developing T cells for negative selection. Mutations in AIRE lead to autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenetic disease. Although it has been reported that AIRE interacts with proteins involved in nuclear transport, DNA-damage response, chromatin remodeling, transcription and pre-mRNA-splicing, the precise mechanism of AIRE-induced tissue restricted antigen expression has remained elusive. In this study, we investigated an APECED patient mutation that causes the loss of the extreme C-terminus of AIRE and found that this mutant protein is transcriptionaly inactive. When tethered heterologously to DNA, this domain could stimulate transcription and splicing by itself. Moreover, the loss of this C-terminus disrupted interactions with the positive transcription elongation factor b (P-TEFb). Via P-TEFb, AIRE increased levels of RNA polymerase II on and enhanced pre-mRNA splicing of heterologous and endogenous target genes. Indeed, the inhibition of CDK9, the kinase subunit of P-TEFb, inhibited AIRE-induced pre-mRNA splicing of these genes. Thus, AIRE requires P-TEFb to activate transcription elongation and co-transcriptional processing of target genes.

  11. Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

    Science.gov (United States)

    Plemenitaš, Ana; Saksela, Kalle; Peterlin, B. Matija

    2011-01-01

    Autoimmune regulator (AIRE) is a transcription factor that induces the expression of a large subset of otherwise strictly tissue restricted antigens in medullary thymic epithelial cells, thereby enabling their presentation to developing T cells for negative selection. Mutations in AIRE lead to autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenetic disease. Although it has been reported that AIRE interacts with proteins involved in nuclear transport, DNA-damage response, chromatin remodeling, transcription and pre-mRNA-splicing, the precise mechanism of AIRE-induced tissue restricted antigen expression has remained elusive. In this study, we investigated an APECED patient mutation that causes the loss of the extreme C-terminus of AIRE and found that this mutant protein is transcriptionaly inactive. When tethered heterologously to DNA, this domain could stimulate transcription and splicing by itself. Moreover, the loss of this C-terminus disrupted interactions with the positive transcription elongation factor b (P-TEFb). Via P-TEFb, AIRE increased levels of RNA polymerase II on and enhanced pre-mRNA splicing of heterologous and endogenous target genes. Indeed, the inhibition of CDK9, the kinase subunit of P-TEFb, inhibited AIRE-induced pre-mRNA splicing of these genes. Thus, AIRE requires P-TEFb to activate transcription elongation and co-transcriptional processing of target genes. PMID:21724609

  12. Putative effects of endocrine disrupters on pubertal development in the human

    DEFF Research Database (Denmark)

    Teilmann, Grete; Juul, Anders; Skakkebaek, Niels E

    2002-01-01

    -called endocrine disrupters. Precocious puberty has been described in several case reports of accidental exposure to oestrogenic compounds in cosmetic products, food and pharmaceuticals. Local epidemics of premature thelarche have also been suggested to be linked to endocrine disrupters. Children adopted from...... developing countries to industrialized countries often develop precocious puberty. Not only precocious puberty, but also delayed puberty can, theoretically, be associated with exposure to endocrine disrupters. While it is very plausible that endocrine disrupters may disturb pubertal development...

  13. Knockout of Foxp2 disrupts vocal development in mice.

    Science.gov (United States)

    Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

    2016-03-16

    The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control.

  14. Association of maternal dopamine transporter genotype with negative parenting: evidence for gene x environment interaction with child disruptive behavior.

    Science.gov (United States)

    Lee, S S; Chronis-Tuscano, A; Keenan, K; Pelham, W E; Loney, J; Van Hulle, C A; Cook, E H; Lahey, B B

    2010-05-01

    Although maternal parenting is central to child development, little is known about the interplay between molecular genetic and environmental factors that influence parenting. We tested the association of the 40-bp variable number tandem repeat polymorphism of the dopamine transporter (DAT1; SLC6A3) gene with three dimensions of observed maternal parenting behavior (positive parenting, negative parenting and total maternal commands). A significant nonadditive association was found between maternal DAT1 genotype and both negative parenting and total commands during a structured mother-child interaction task, even after controlling demographic factors, maternal psychopathology and disruptive child behavior during the task. Furthermore, the association between maternal DAT1 genotype and negative parenting was significantly stronger among mothers whose children were highly disruptive during the mother-child interaction task, suggesting a gene-environment interaction.

  15. Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

    OpenAIRE

    Zhang, Dong-Xiu; Nuss, Donald L.

    2016-01-01

    Allorecognition, termed “vegetative incompatibility” (vic), in filamentous fungi limits the efficacy of virulence-attenuating mycoviruses for biological control (hypovirulence). Because mycoviruses lead exclusive intracellular lifestyles, horizontal transmission relies on cytoplasmic exchange during hyphal fusion (anastomosis). Fusion between vic-incompatible strains results in localized cell death restricting mycovirus transmission. We report the systematic disruption of multilocus vic genes...

  16. New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe

    OpenAIRE

    Gadaleta, Mariana C.; Iwasaki, Osamu; Noguchi, Chiaki; Noma, Ken-ichi; Noguchi, Eishi

    2013-01-01

    We describe a series of new vectors for PCR-based epitope tagging and gene disruption in the fission yeast Schizosaccharomyces pombe, an exceptional model organism for the study of cellular processes. The vectors are designed for amplification of gene-targeting DNA cassettes and integration into specific genetic loci, allowing expression of proteins fused to 12 tandem copies of the Pk (V5) epitope or 5 tandem copies of the FLAG epitope with a glycine linker. These vectors are available with v...

  17. Slc4a11 Gene Disruption in Mice

    Science.gov (United States)

    Lopez, Ivan A.; Rosenblatt, Mark I.; Kim, Charles; Galbraith, Gary C.; Jones, Sherri M.; Kao, Liyo; Newman, Debra; Liu, Weixin; Yeh, Stacey; Pushkin, Alexander; Abuladze, Natalia; Kurtz, Ira

    2009-01-01

    NaBC1 (the SLC4A11 gene) belongs to the SLC4 family of sodium-coupled bicarbonate (carbonate) transporter proteins and functions as an electrogenic sodium borate cotransporter. Mutations in SLC4A11 cause either corneal abnormalities (corneal hereditary dystrophy type 2) or a combined auditory and visual impairment (Harboyan syndrome). The role of NaBC1 in sensory systems is poorly understood, given the difficulty of studying patients with NaBC1 mutations. We report our findings in Slc4a11−/− mice generated to investigate the role of NaBC1 in sensorineural systems. In wild-type mice, specific NaBC1 immunoreactivity was detected in fibrocytes of the spiral ligament, from the basal to the apical portion of the cochlea. NaBC1 immunoreactivity was present in the vestibular labyrinth, in stromal cells underneath the non-immunoreactive sensory epithelia of the macula utricle, sacule, and crista ampullaris, and the membranous vestibular labyrinth was collapsed. Both auditory brain response and vestibular evoked potential waveforms were significantly abnormal in Slc4a11−/− mice. In the cornea, NaBC1 was highly expressed in the endothelial cell layer with less staining in epithelial cells. However, unlike humans, the corneal phenotype was mild with a normal slit lamp evaluation. Corneal endothelial cells were morphologically normal; however, both the absolute height of the corneal basal epithelial cells and the relative basal epithelial cell/total corneal thickness were significantly increased in Slc4a11−/− mice. Our results demonstrate for the first time the importance of NaBC1 in the audio-vestibular system and provide support for the hypothesis that SLC4A11 should be considered a potential candidate gene in patients with isolated sensorineural vestibular hearing abnormalities. PMID:19586905

  18. Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Arikawa, Y; Kuroyanagi, T; Shimosaka, M; Muratsubaki, H; Enomoto, K; Kodaira, R; Okazaki, M

    1999-01-01

    Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.

  19. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

    Directory of Open Access Journals (Sweden)

    Vita M. Golubovskaya

    2014-01-01

    Full Text Available Focal Adhesion Kinase (FAK is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53+/+ and p53−/− cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05 in HCT116 p53+/+ cells but not in p53−/− cells. Among up-regulated genes in HCT p53+/+ cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53+/+ colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  20. New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe.

    Science.gov (United States)

    Gadaleta, Mariana C; Iwasaki, Osamu; Noguchi, Chiaki; Noma, Ken-ichi; Noguchi, Eishi

    2013-11-01

    We describe a series of new vectors for PCR-based epitope tagging and gene disruption in the fission yeast Schizosaccharomyces pombe, an exceptional model organism for the study of cellular processes. The vectors are designed for amplification of gene-targeting DNA cassettes and integration into specific genetic loci, allowing expression of proteins fused to 12 tandem copies of the Pk (V5) epitope or 5 tandem copies of the FLAG epitope with a glycine linker. These vectors are available with various antibiotic or nutritional markers and are useful for protein studies using biochemical and cell biological methods. We also describe new vectors for fluorescent protein-tagging and gene disruption using ura4MX6, LEU2MX6, and his3MX6 selection markers, allowing researchers in the S. pombe community to disrupt genes and manipulate genomic loci using primer sets already available for the widely used pFA6a-MX6 system. Our new vectors may also be useful for gene manipulation in Saccharomyces cerevisiae.

  1. α-Mangostin disrupts the development of Streptococcus mutans biofilms and facilitates its mechanical removal.

    Science.gov (United States)

    Nguyen, Phuong Thi Mai; Falsetta, Megan L; Hwang, Geelsu; Gonzalez-Begne, Mireya; Koo, Hyun

    2014-01-01

    α-Mangostin (αMG) has been reported to be an effective antimicrobial agent against planktonic cells of Streptococcus mutans, a biofilm-forming and acid-producing cariogenic organism. However, its anti-biofilm activity remains to be determined. We examined whether αMG, a xanthone purified from Garcinia mangostana L grown in Vietnam, disrupts the development, acidogenicity, and/or the mechanical stability of S. mutans biofilms. Treatment regimens simulating those experienced clinically (twice-daily, 60 s exposure each) were used to assess the bioactivity of αMG using a saliva-coated hydroxyapatite (sHA) biofilm model. Topical applications of early-formed biofilms with αMG (150 µM) effectively reduced further biomass accumulation and disrupted the 3D architecture of S. mutans biofilms. Biofilms treated with αMG had lower amounts of extracellular insoluble and intracellular iodophilic polysaccharides (30-45%) than those treated with vehicle control (P3-fold biofilm detachment from sHA vs. vehicle-treated biofilms; PgtfC genes was unaffected. Hence, this study reveals that brief exposures to αMG can disrupt the development and structural integrity of S. mutans biofilms, at least in part via inhibition of key enzymatic systems associated with exopolysaccharide synthesis and acidogenicity. αMG could be an effective anti-virulence additive for the control and/or removal of cariogenic biofilms.

  2. Gene expression disruptions of organism versus organ in Drosophila species hybrids.

    Directory of Open Access Journals (Sweden)

    Daniel J Catron

    2008-08-01

    Full Text Available Hybrid dysfunctions, such as sterility, may result in part from disruptions in the regulation of gene expression. Studies of hybrids within the Drosophila simulans clade have reported genes expressed above or below the expression observed in their parent species, and such misexpression is associated with male sterility in multigenerational backcross hybrids. However, these studies often examined whole bodies rather than testes or had limited replication using less-sensitive but global techniques. Here, we use a new RNA isolation technique to re-examine hybrid gene expression disruptions in both testes and whole bodies from single Drosophila males by real-time quantitative RT-PCR. We find two early-spermatogenesis transcripts are underexpressed in hybrid whole-bodies but not in assays of testes alone, while two late-spermatogenesis transcripts seem to be underexpressed in both whole-bodies and testes alone. Although the number of transcripts surveyed is limited, these results provide some support for a previous hypothesis that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes.

  3. HDAC Inhibitors Disrupt Programmed Resistance to Apoptosis During Drosophila Development

    Directory of Open Access Journals (Sweden)

    Yunsik Kang

    2017-06-01

    Full Text Available We have previously shown that the ability to respond to apoptotic triggers is regulated during Drosophila development, effectively dividing the fly life cycle into stages that are either sensitive or resistant to apoptosis. Here, we show that the developmentally programmed resistance to apoptosis involves transcriptional repression of critical proapoptotic genes by histone deacetylases (HDACs. Administration of HDAC inhibitors (HDACi, like trichostatin A or suberoylanilide hydroxamic acid, increases expression of proapoptotic genes and is sufficient to sensitize otherwise resistant stages. Conversely, reducing levels of proapoptotic genes confers resistance to otherwise sensitive stages. Given that resistance to apoptosis is a hallmark of cancer cells, and that HDACi have been recently added to the repertoire of FDA-approved agents for cancer therapy, our results provide new insights for how HDACi help kill malignant cells and also raise concerns for their potential unintended effects on healthy cells.

  4. Strategy development for anticipating and handling a disruptive technology.

    Science.gov (United States)

    Chan, Stephen

    2006-10-01

    The profession of radiology has greatly benefited from the introduction of new imaging technologies throughout its history. Therefore, it would seem reasonable for radiologists to believe that the emergence of a new imaging technology can generally be foreseen with sufficient advance notice to allow the appropriate levels of time, effort, and money to be devoted toward incorporating it into radiology practice. However, in his seminal work, Christiansen characterized a new form of technologic innovation, known as "disruptive technology," whose emergence often heralds the replacement of market leaders in an industry by competitors who are quicker in adopting and deploying the new technology. This article briefly describes the phenomenon of disruptive technology and addresses the challenges that organizations face in dealing with disruptive technology. The article raises 4 questions about the future of radiology: (1) Are health care and radiology vulnerable to disruptive technology? (2) What kinds of change may be in store for the radiology profession? (3) Can the radiology profession prepare itself to recognize and respond to a disruptive innovation among a group of new imaging technologies? and (4) How should a radiology organization decide whether to invest significant resources in a potentially disruptive technology? This article addresses these questions by reviewing key insights from leading "gurus" in the fields of competitive strategy and technology management and applying them to radiology. This illustrates how and why (despite past successes) the radiology profession may still have a blind spot in recognizing and handling disruptive technologies.

  5. Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes.

    Science.gov (United States)

    Zhang, Dong-Xiu; Nuss, Donald L

    2016-02-23

    Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

  6. Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

    Science.gov (United States)

    Abrahimi, Parwiz; Chang, William G; Kluger, Martin S; Qyang, Yibing; Tellides, George; Saltzman, W Mark; Pober, Jordan S

    2015-07-03

    The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering. © 2015 American Heart Association, Inc.

  7. Gene Expression Profiling Identifies Important Genes Affected by R2 Compound Disrupting FAK and P53 Complex

    Energy Technology Data Exchange (ETDEWEB)

    Golubovskaya, Vita M., E-mail: Vita.Golubovskaya@roswellpark.org; Ho, Baotran [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Conroy, Jeffrey [Genomics Shared Resource, Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Liu, Song; Wang, Dan [Bioinformatics Core Facility, Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States); Cance, William G. [Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263 (United States)

    2014-01-21

    Focal Adhesion Kinase (FAK) is a non-receptor kinase that plays an important role in many cellular processes: adhesion, proliferation, invasion, angiogenesis, metastasis and survival. Recently, we have shown that Roslin 2 or R2 (1-benzyl-15,3,5,7-tetraazatricyclo[3.3.1.1~3,7~]decane) compound disrupts FAK and p53 proteins, activates p53 transcriptional activity, and blocks tumor growth. In this report we performed a microarray gene expression analysis of R2-treated HCT116 p53{sup +/+} and p53{sup −/−} cells and detected 1484 genes that were significantly up- or down-regulated (p < 0.05) in HCT116 p53{sup +/+} cells but not in p53{sup −/−} cells. Among up-regulated genes in HCT p53{sup +/+} cells we detected critical p53 targets: Mdm-2, Noxa-1, and RIP1. Among down-regulated genes, Met, PLK2, KIF14, BIRC2 and other genes were identified. In addition, a combination of R2 compound with M13 compound that disrupts FAK and Mmd-2 complex or R2 and Nutlin-1 that disrupts Mdm-2 and p53 decreased clonogenicity of HCT116 p53{sup +/+} colon cancer cells more significantly than each agent alone in a p53-dependent manner. Thus, the report detects gene expression profile in response to R2 treatment and demonstrates that the combination of drugs targeting FAK, Mdm-2, and p53 can be a novel therapy approach.

  8. CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

    Science.gov (United States)

    Billon, Pierre; Bryant, Eric E; Joseph, Sarah A; Nambiar, Tarun S; Hayward, Samuel B; Rothstein, Rodney; Ciccia, Alberto

    2017-09-21

    Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Efficient gene replacements in ku70 disruption strain of Aspergillus chevalieri var. intermedius

    Directory of Open Access Journals (Sweden)

    Qingqing Huang

    2017-01-01

    Full Text Available Aspergillus chevalieri var. intermedius is a dominant filamentous fungal species in Fuzhuan tea and is associated with the quality and health benefits of this tea. The sexual or asexual reproduction of this fungus depends on the osmotic pressure of the tea. Efforts to enhance the beneficial effects of A. chevalieri var. intermedius are hampered by difficulties in disrupting its genes. To address this issue, we identified the A. chevalieri var. intermedius homolog (Acku70 of human Ku70 and generated an Acku70 disruption strain (ΔAcku70, aiming to improve the gene replacement efficiency. ΔAcku70 grew at a slightly lower rate in vitro than the wild-type strain; however, the two strains exhibited similar sensitivity to temperature, osmotic pressure and the effects of ethyl methane sulphonate and H2O2. The replacement efficiency of veA and flbA dramatically increased in ΔAcku70 compared to that in the wild type. The efficiency of flbA replacement increased from 2.6% to 80%, whereas the frequency of veA disruption increased from 15.2% to 83.9% and from 30.8% to 86.8%. Thus, ΔAcku70 is suitable for use as a type strain for large-scale functional genomic analysis of A. chevalieri var. intermedius.

  10. Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli.

    Science.gov (United States)

    Imaizumi, Akira; Takikawa, Rie; Koseki, Chie; Usuda, Yoshihiro; Yasueda, Hisashi; Kojima, Hiroyuki; Matsui, Kazuhiko; Sugimoto, Shin-Ichi

    2005-04-20

    Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.

  11. Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions

    Directory of Open Access Journals (Sweden)

    Guo Yanhai

    2012-10-01

    Full Text Available Abstract Background The core protein (HBc of hepatitis B virus (HBV has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression. Results Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC, type 1 insulin-like growth factor receptor (IGF1R, and neurotrophic tyrosine kinase receptor 2 (NTRK2, and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS. Conclusion HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

  12. Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

    OpenAIRE

    Driessen, Carola A. G. G.; Winkens, Huub J.; Hoffmann, Kirstin; Kuhlmann, Leonoor D.; Janssen, Bert P. M.; Van Vugt, Anke H. M.; Van Hooser, J. Preston; Wieringa, B. E.; Deutman, August F; Palczewski, Krzysztof; Ruether, Klaus; Janssen, Jacques J. M.

    2000-01-01

    To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol deh...

  13. Examining gene-environment interactions in comorbid depressive and disruptive behavior disorders using a Bayesian approach.

    Science.gov (United States)

    Adrian, Molly; Kiff, Cara; Glazner, Chris; Kohen, Ruth; Tracy, Julia Helen; Zhou, Chuan; McCauley, Elizabeth; Vander Stoep, Ann

    2015-09-01

    The objective of this study was to apply a Bayesian statistical analytic approach that minimizes multiple testing problems to explore the combined effects of chronic low familial support and variants in 12 candidate genes on risk for a common and debilitating childhood mental health condition. Bayesian mixture modeling was used to examine gene by environment interactions among genetic variants and environmental factors (family support) associated in previous studies with the occurrence of comorbid depression and disruptive behavior disorders youth, using a sample of 255 children. One main effect, variants in the oxytocin receptor (OXTR, rs53576) was associated with increased risk for comorbid disorders. Two significant gene × environment and one signification gene × gene interactions emerged. Variants in the nicotinic acetylcholine receptor α5 subunit (CHRNA5, rs16969968) and in the glucocorticoid receptor chaperone protein FK506 binding protein 5 (FKBP5, rs4713902) interacted with chronic low family support in association with child mental health status. One gene × gene interaction, 5-HTTLPR variant of the serotonin transporter (SERT/SLC6A4) in combination with μ opioid receptor (OPRM1, rs1799971) was associated with comorbid depression and conduct problems. Results indicate that Bayesian modeling is a feasible strategy for conducting behavioral genetics research. This approach, combined with an optimized genetic selection strategy (Vrieze et al., 2012), revealed genetic variants involved in stress regulation (FKBP5, SERT × OPMR), social bonding (OXTR), and nicotine responsivity (CHRNA5) in predicting comorbid status. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Examining Gene-Environment Interactions in Comorbid Depressive and Disruptive Behavior Disorders using a Bayesian Approach

    Science.gov (United States)

    Adrian, Molly; Kiff, Cara; Glazner, Chris; Kohen, Ruth; Tracy, Julia Helen; Zhou, Chuan; McCauley, Elizabeth; Stoep, Ann Vander

    2015-01-01

    Objective The objective of this study was to apply a Bayesian statistical analytic approach that minimizes multiple testing problems to explore the combined effects of chronic low familial support and variants in 12 candidate genes on risk for a common and debilitating childhood mental health condition. Method Bayesian mixture modeling was used to examine gene by environment interactions among genetic variants and environmental factors (family support) associated in previous studies with the occurrence of comorbid depression and disruptive behavior disorders youth, using a sample of 255 children. Results One main effects, variants in the oxytocin receptor (OXTR, rs53576) was associated with increased risk for comorbid disorders. Two significant gene x environment and one signification gene x gene interaction emerged. Variants in the nicotinic acetylcholine receptor α5 subunit (CHRNA5, rs16969968) and in the glucocorticoid receptor chaperone protein FK506 binding protein 5 (FKBP5, rs4713902) interacted with chronic low family support in association with child mental health status. One gene x gene interaction, 5-HTTLPR variant of the serotonin transporter (SERT/SLC6A4) in combination with μ opioid receptor (OPRM1, rs1799971) was associated with comorbid depression and conduct problems. Conclusions Results indicate that Bayesian modeling is a feasible strategy for conducting behavioral genetics research. This approach, combined with an optimized genetic selection strategy (Vrieze, Iacono, & McGue, 2012), revealed genetic variants involved in stress regulation ( FKBP5, SERTxOPMR), social bonding (OXTR), and nicotine responsivity (CHRNA5) in predicting comorbid status. PMID:26228411

  15. High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library

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    Schulte Julia

    2002-07-01

    Full Text Available Abstract Background The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. Results As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2% that differed from the wild-type in a variety of developmental, morphological and physiological characteristics. Conclusions The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

  16. Disruption of the homogentisate solanesyltransferase gene results in albino and dwarf phenotypes and root, trichome and stomata defects in Arabidopsis thaliana.

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    Yuehui Chao

    Full Text Available Homogentisate solanesyltransferase (HST plays an important role in plastoquinone (PQ biosynthesis and acts as the electron acceptor in the carotenoids and abscisic acid (ABA biosynthesis pathways. We isolated and identified a T-DNA insertion mutant of the HST gene that displayed the albino and dwarf phenotypes. PCR analyses and functional complementation also confirmed that the mutant phenotypes were caused by disruption of the HST gene. The mutants also had some developmental defects, including trichome development and stomata closure defects. Chloroplast development was also arrested and chlorophyll (Chl was almost absent. Developmental defects in the chloroplasts were consistent with the SDS-PAGE result and the RNAi transgenic phenotype. Exogenous gibberellin (GA could partially rescue the dwarf phenotype and the root development defects and exogenous ABA could rescue the stomata closure defects. Further analysis showed that ABA and GA levels were both very low in the pds2-1 mutants, which suggested that biosynthesis inhibition by GAs and ABA contributed to the pds2-1 mutants' phenotypes. An early flowering phenotype was found in pds2-1 mutants, which showed that disruption of the HST gene promoted flowering by partially regulating plant hormones. RNA-sequencing showed that disruption of the HST gene resulted in expression changes to many of the genes involved in flowering time regulation and in the biosynthesis of PQ, Chl, GAs, ABA and carotenoids. These results suggest that HST is essential for chloroplast development, hormone biosynthesis, pigment accumulation and plant development.

  17. Parasexual genetics of Dictyostelium gene disruptions: identification of a ras pathway using diploids

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    Insall Robert H

    2003-07-01

    Full Text Available Abstract Background The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes. Results We describe new techniques for parasexual fusion of strains in liquid medium, selection and maintenance of the resulting stable diploid strains, and segregation to make recombined haploids. We have used these techniques to isolate rasS/gefB double nulls. The phenotypes of these mutants are no more severe than either parent, with movement, phagocytosis and fluid-phase endocytosis affected to the same degree as in rasS or gefB single nulls. In addition, we have produced diploids from one AX2- and one AX3-derived parent, providing an axenic strain with fewer secondary phenotypes than has been previously available. Conclusions The phenotype of the rasS/gefB double mutant suggests that the RasS and GefB proteins lie on the same linear pathway. In addition, axenic diploids and the techniques to generate, maintain and segregate them will be productive tools for future work on Dictyostelium. They will particularly facilitate generation of multiple mutants and manuipulation of essential genes.

  18. Mfsd14a (Hiat1) gene disruption causes globozoospermia and infertility in male mice.

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    Doran, Joanne; Walters, Cara; Kyle, Victoria; Wooding, Peter; Hammett-Burke, Rebecca; Colledge, William Henry

    2016-07-01

    The Mfsd14a gene, previously called Hiat1, encodes a transmembrane protein of unknown function with homology to the solute carrier protein family. To study the function of the MFSD14A protein, mutant mice (Mus musculus, strain 129S6Sv/Ev) were generated with the Mfsd14a gene disrupted with a LacZ reporter gene. Homozygous mutant mice are viable and healthy, but males are sterile due to a 100-fold reduction in the number of spermatozoa in the vas deferens. Male mice have adequate levels of testosterone and show normal copulatory behaviour. The few spermatozoa that are formed show rounded head defects similar to those found in humans with globozoospermia. Spermatogenesis proceeds normally up to the round spermatid stage, but the subsequent structural changes associated with spermiogenesis are severely disrupted with failure of acrosome formation, sperm head condensation and mitochondrial localization to the mid-piece of the sperm. Staining for β-galactosidase activity as a surrogate for Mfsd14a expression indicates expression in Sertoli cells, suggesting that MFSD14A may transport a solute from the bloodstream that is required for spermiogenesis. © 2016 Society for Reproduction and Fertility.

  19. Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

    Science.gov (United States)

    Shen, Bang; Brown, Kevin M.; Lee, Tobie D.

    2014-01-01

    ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. PMID:24825012

  20. Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9.

    Science.gov (United States)

    Shen, Bang; Brown, Kevin M; Lee, Tobie D; Sibley, L David

    2014-05-13

    Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. However, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to such approaches. Unfortunately, such common lab isolates have also been passaged frequently in vitro and have thus lost many of the

  1. Dysphagia and disrupted cranial nerve development in a mouse model of DiGeorge (22q11 deletion syndrome

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    Beverly A. Karpinski

    2014-02-01

    Full Text Available We assessed feeding-related developmental anomalies in the LgDel mouse model of chromosome 22q11 deletion syndrome (22q11DS, a common developmental disorder that frequently includes perinatal dysphagia – debilitating feeding, swallowing and nutrition difficulties from birth onward – within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA signaling, prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V, glossopharyngeal (IX or vagus (X cranial nerves (CNs that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to those in wild-type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and CN development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS.

  2. Dysphagia and disrupted cranial nerve development in a mouse model of DiGeorge (22q11) deletion syndrome

    Science.gov (United States)

    Karpinski, Beverly A.; Maynard, Thomas M.; Fralish, Matthew S.; Nuwayhid, Samer; Zohn, Irene E.; Moody, Sally A.; LaMantia, Anthony-S.

    2014-01-01

    ABSTRACT We assessed feeding-related developmental anomalies in the LgDel mouse model of chromosome 22q11 deletion syndrome (22q11DS), a common developmental disorder that frequently includes perinatal dysphagia – debilitating feeding, swallowing and nutrition difficulties from birth onward – within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA) signaling, prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V), glossopharyngeal (IX) or vagus (X) cranial nerves (CNs) that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to those in wild-type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and CN development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS. PMID:24357327

  3. Autophagy Genes of Host Responds to Disruption of Gut Microbial Community by Antibiotics.

    Science.gov (United States)

    Singh, Sudha B; Wilson, Melissa; Ritz, Nathaniel; Lin, Henry C

    2017-06-01

    Defective autophagic machinery, such as that in Crohn's disease patients homozygous for ATG16L1 risk allele, is associated with alteration of resident gut bacterial communities. However, whether or not host autophagy responds to changes in the resident gut microbial community is not known. Here, we investigated the effect of antibiotic-induced disruption of the gut microbiome (dysbiosis) on autophagy gene expression and the expression of antimicrobial peptides/protein (AMP) over time. To test the hypothesis that antibiotic treatment may cause time-dependent changes in gut bacterial density, autophagy genes, and antimicrobial protein/peptide gene expression. Mice (n = 8 per group) were treated with antibiotic cocktail and sacrificed at different intervals of recovery (days 3, 7, 10, 14, 21, 28, 35, and 42) post-antibiotics. DNA and RNA were extracted from small intestinal tissues. Bacterial density, expression of host autophagy genes, and AMP genes were analyzed by relative quantitative PCR. Fold change difference in comparison with untreated control group was calculated using 2 -ΔΔCt method. Statistical analysis was performed using nonparametric Mann-Whitney test. Gut bacterial density changed in a time-dependent fashion in response to antibiotic treatment. These changes were concurrent with upregulation of autophagy genes and antimicrobial peptide/protein gene expression. We further showed that an oral gavage of a resident microbe Desulfovibrio, which bloomed in antibiotic-treated animals, induced Atg5 and lysozyme (Lyz) gene expression. Autophagy genes respond to dysbiosis induced by antibiotics. This response may be a host mechanism to detect and possibly correct dysbiosis by activating antimicrobial peptides/proteins that control the microbial load in the gut.

  4. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

    Science.gov (United States)

    Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

    2015-10-01

    Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line

  5. Disruptive CHD8 mutations define a subtype of autism early in development

    Science.gov (United States)

    Bernier, Raphael; Golzio, Christelle; Xiong, Bo; Stessman, Holly; Coe, Bradley P.; Penn, Osnat; Witherspoon, Kali; Gerdts, Jennifer; Baker, Carl; Vulto-van Silfhout, Anneke T.; Schuurs-Hoeijmakers, Janneke H.; Fichera, Marco; Bosco, Paolo; Buono, Serafino; Alberti, Antonino; Failla, Pinella; Peeters, Hilde; Steyaert, Jean; Vissers, Lisenka E.L.M.; Francescatto, Ludmila; Mefford, Heather C.; Rosenfeld, Jill A.; Bakken, Trygve; O’Roak, Brian J.; Pawlus, Matthew; Moon, Randall; Shendure, Jay; Amaral, David G.; Lein, Ed; Rankin, Julia; Romano, Corrado; de Vries, Bert B.A.; Katsanis, Nicholas; Eichler, Evan E.

    2014-01-01

    Autism spectrum disorder (ASD) is a heterogeneous disease where efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in post-mitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation. PMID:24998929

  6. The growth hormone receptor gene-disrupted mouse fails to respond to an intermittent fasting diet.

    Science.gov (United States)

    Arum, Oge; Bonkowski, Michael S; Rocha, Juliana S; Bartke, Andrzej

    2009-12-01

    The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long lived; and this longevity is not responsive to 30% caloric restriction, in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction, we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting diet. The intermittent fasting diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction, we postulate that GHR-KO mice would be resistant to any manner of dietary restriction; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan extending potential of an intervention.

  7. The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells.

    Science.gov (United States)

    Khosla, Meenal; Spiegelman, George B; Weeks, Gerald

    2005-08-15

    The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.

  8. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants.

    Science.gov (United States)

    Valdés-Santiago, Laura; Guzmán-de-Peña, Doralinda; Ruiz-Herrera, José

    2010-11-01

    In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

  9. α-Mangostin disrupts the development of Streptococcus mutans biofilms and facilitates its mechanical removal.

    Directory of Open Access Journals (Sweden)

    Phuong Thi Mai Nguyen

    Full Text Available α-Mangostin (αMG has been reported to be an effective antimicrobial agent against planktonic cells of Streptococcus mutans, a biofilm-forming and acid-producing cariogenic organism. However, its anti-biofilm activity remains to be determined. We examined whether αMG, a xanthone purified from Garcinia mangostana L grown in Vietnam, disrupts the development, acidogenicity, and/or the mechanical stability of S. mutans biofilms. Treatment regimens simulating those experienced clinically (twice-daily, 60 s exposure each were used to assess the bioactivity of αMG using a saliva-coated hydroxyapatite (sHA biofilm model. Topical applications of early-formed biofilms with αMG (150 µM effectively reduced further biomass accumulation and disrupted the 3D architecture of S. mutans biofilms. Biofilms treated with αMG had lower amounts of extracellular insoluble and intracellular iodophilic polysaccharides (30-45% than those treated with vehicle control (P3-fold biofilm detachment from sHA vs. vehicle-treated biofilms; P<0.05. Moreover, acid production by S. mutans biofilms was disrupted following αMG treatments (vs. vehicle-control, P<0.05. The activity of enzymes associated with glucan synthesis, acid production, and acid tolerance (glucosyltransferases B and C, phosphotransferase-PTS system, and F1F0-ATPase were significantly inhibited by αMG. The expression of manL, encoding a key component of the mannose PTS, and gtfB were slightly repressed by αMG treatment (P<0.05, while the expression of atpD (encoding F-ATPase and gtfC genes was unaffected. Hence, this study reveals that brief exposures to αMG can disrupt the development and structural integrity of S. mutans biofilms, at least in part via inhibition of key enzymatic systems associated with exopolysaccharide synthesis and acidogenicity. αMG could be an effective anti-virulence additive for the control and/or removal of cariogenic biofilms.

  10. Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development.

    Science.gov (United States)

    Magie, C R; Meyer, M R; Gorsuch, M S; Parkhurst, S M

    1999-12-01

    Rho GTPases play an important role in diverse biological processes such as actin cytoskeleton organization, gene transcription, cell cycle progression and adhesion. They are required during early Drosophila development for proper execution of morphogenetic movements of individual cells and groups of cells important for the formation of the embryonic body plan. We isolated loss-of-function mutations in the Drosophila Rho1 (Rho1) gene during a genetic screen for maternal-effect mutations, allowing us to investigate the specific roles Rho1 plays in the context of the developing organism. Here we report that Rho1 is required for many early events: loss of Rho1 function results in both maternal and embryonic phenotypes. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Two phenotypes are associated with reduction of maternal Rho1 activity: the actin cytoskeleton is disrupted in egg chambers, especially in the ring canals and embryos display patterning defects as a result of improper maintenance of segmentation gene expression. Despite showing imperfect dorsal closure, Rho1 does not activate downstream genes or interact genetically with members of the JNK signaling pathway, used by its relatives dRac and dCdc42 for proper dorsal closure. Consistent with its roles in regulating actin cytoskeletal organization, we find that Rho1 interacts genetically and physically with the Drosophila formin homologue, cappuccino. We also show that Rho1 interacts both genetically and physically with concertina, a G(alpha) protein involved in cell shape changes during gastrulation.

  11. Development of an economic framework to evaluate resilience in recovering from major port disruptions : final report.

    Science.gov (United States)

    2017-02-03

    Ports play a critical role in a nations economic system. The impact of a major port disruption can reverberate across the entire economy through regional and national supply-chains. This study develops an operational framework to evaluate the effe...

  12. Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hyejin; Yoon, Min-Sik; Ryu, Kwon-Yul, E-mail: kyryu@uos.ac.kr

    2013-06-07

    Highlights: •Proliferation capacity of Ubc{sup −/−} FLCs was reduced during culture in vitro. •Ubc is required for proliferation of both hepatocytes and bipotent FLEPCs. •Bipotent FLEPCs exhibit highest Ubc transcription and proliferation capacity. •Cell types responsible for Ubc{sup −/−} fetal liver developmental defect were identified. -- Abstract: We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc{sup −/−} embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc{sup −/−} cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid

  13. Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ida, Yoshihiro; Furusawa, Chikara; Hirasawa, Takashi; Shimizu, Hiroshi

    2012-02-01

    We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, Navdeep [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Vijayan, Mathilakath M., E-mail: mvijayan@uwaterloo.ca [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada)

    2011-05-15

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  15. Time for a New Agenda: Organizational Development in a Changing world with much Disruption

    DEFF Research Database (Denmark)

    Sørensen, Henrik B.

    2017-01-01

    organizations must be more flexible and adaptive and must improve their communication with the community and the crowd of the company, as this is a prerequisite for a company in a disruptive world. When companies use the traditional development path only, their main focus is on exploiting existing possibilities...... demands for bureaucracy, but which at the same time, via its organic organizational structure, includes the external demands of today’s knowledge society, i.e. exploring new opportunities. The answer to this research question will primarily be sought in the theories of organizational evolution...... organizations neglect to support a disruptive strategy. By demonstrating the existence of another development path, this paper attempts, from a theoretical point of view, to give a new and a more nuanced perspective on organizational development in a disruptive world. This new path is supportive in a disruptive...

  16. Targeted disruption of the spermatid-specific gene Spata31 causes male infertility.

    Science.gov (United States)

    Wu, Yuan-Yi; Yang, Yong; Xu, Yong-De; Yu, Hua-Liang

    2015-06-01

    Spata31, a novel testis-specific gene, was first isolated from the testis of a vitamin A-deficient rat model. To gain insight into its physiological function, Spata31-targeted knockout mice were generated by homologous recombination. Spata31-deficient (Spata31(flox/flox) ; Vasa-Cre) male mice exhibited low sperm count and premature shedding of germ cells into the lumen, ultimately causing azoospermia and male sterility. Mechanistically, the Spata31 deficiency resulted in reduced expression of the adhesion protein nectin-3 and cytoskeletal protein β-actin at the apical ectoplasmic specialization. Our findings demonstrate that the disruptions to the SPATA31 ortholog could be linked to human male infertility. © 2015 Wiley Periodicals, Inc.

  17. Trypanosoma cruzi: attenuation of virulence and protective immunogenicity after monoallelic disruption of the cub gene.

    Science.gov (United States)

    Barrio, Alejandra B; Van Voorhis, Wesley C; Basombrío, Miguel A

    2007-12-01

    Calmodulin-ubiquitin (cub) is a single-copy gene of Trypanosoma cruzi, which encodes a 208 aminoacid polypeptide of unknown function, containing putative calcium-binding domains. After targeted deletion, a clone (TulCub8) was derived where one of the two alleles was disrupted. This clone displayed a sharp and stable loss of virulence for mice. Parasitemias after inoculation of 10(6) trypomastigotes of the mutant, as compared to wild-type parasites were 68-fold lower (p=0.018) in adult Swiss mice and 27-fold lower (p=0.002) in newborn Balb/c mice. Epimastigote inocula of the mutant were strongly protective against infection by wild-type parasites. Virulence was not restored by serial passage in mice, showing that the attenuated phenotype is stable and gene-conversion from the intact cub allele does not occur at an appreciable rate. Retransfection of the missing cub allele restored virulence. Complementation experiments showed that the intact cub gene is necessary for full expression of virulence.

  18. Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis.

    Science.gov (United States)

    Reichard, U; Hung, C Y; Thomas, P W; Cole, G T

    2000-10-01

    Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss of CTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Deltacts1 strains suggested that the absence of a functional CTS1 gene can be compensated for by elevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Deltacts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

  19. Methylmercury-induced changes in gene transcription associated with neuroendocrine disruption in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Richter, Catherine A.; Martyniuk, Christopher J.; Annis, Mandy L.; Brumbaugh, William G.; Chasar, Lia C.; Denslow, Nancy D.; Tillitt, Donald E.

    2014-01-01

    Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 μg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

  20. Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

    Science.gov (United States)

    2013-01-01

    Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

  1. The relationship between circadian disruption and the development of metabolic syndrome and type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Karatsoreos IN

    2014-12-01

    Full Text Available Ilia N Karatsoreos Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA Abstract: Circadian (daily rhythms are pervasive in nature, and expressed in nearly every behavioral and physiological process. In mammals, circadian rhythms are regulated by the master brain clock in the suprachiasmatic nucleus of the hypothalamus that coordinates the activity of “peripheral” oscillators throughout the brain and body. While much progress has been made in understanding the basic functioning of the circadian clock at the level of genes, molecules, and cells, our understanding of how these clocks interact with complex systems is still in its infancy. Much recent work has focused on the role of circadian clocks in the etiology of disorders as diverse as cancer, diabetes, and obesity. Given the rapid rise in obesity, and the economic costs involved in treating its associated cardiometabolic disorders such as heart disease and diabetes mellitus, understanding the development of obesity and metabolic dysregulation is crucial. Significant epidemiological data indicate a role for circadian rhythms in metabolic disorders. Shift workers have a higher incidence of obesity and diabetes, and laboratory studies in humans show misaligning sleep and the circadian clock leads to hyperinsulinemia. In animal models, body-wide “clock gene” knockout mice are prone to obesity. Further, disrupting the circadian clock by manipulating the light–dark cycle can result in metabolic dysregulation and development of obesity. At the molecular level, elegant studies have shown that targeted disruption of the genetic circadian clock in the pancreas leads to diabetes, highlighting the fact that the circadian clock is directly coupled to metabolism at the cellular level. Keywords: glucose, metabolism, sleep, rhythms, obesity

  2. Hypothalamus region-specific global gene expression profiling in early stages of central endocrine disruption in rat neonates injected with estradiol benzoate or flutamide

    National Research Council Canada - National Science Library

    Shibutani, Makoto; Lee, Kyoung-Youl; Igarashi, Katsuhide; Woo, Gye-Hyeong; Inoue, Kaoru; Nishimura, Tetsuji; Hirose, Masao

    2007-01-01

    To identify genes linked to early stages of disruption of brain sexual differentiation, hypothalamic region-specific microarray analyses were performed using a microdissection technique with neonatal...

  3. Disrupted social development enhances the motivation for cocaine in rats

    NARCIS (Netherlands)

    Baarendse, P.J.J.; Limpens, J.H.W.; Vanderschuren, L.J.M.J.|info:eu-repo/dai/nl/126514917

    2014-01-01

    for behavioural development. In particular, social play behaviour during post-weaning development is thought to facilitate the attainment of social, emotional and cognitive capacities. Conversely, social insults during development can cause longlasting behavioural impairments and increase the

  4. Fundamental device design considerations in the development of disruptive nanoelectronics.

    Science.gov (United States)

    Singh, R; Poole, J O; Poole, K F; Vaidya, S D

    2002-01-01

    In the last quarter of a century silicon-based integrated circuits (ICs) have played a major role in the growth of the economy throughout the world. A number of new technologies, such as quantum computing, molecular computing, DNA molecules for computing, etc., are currently being explored to create a product to replace semiconductor transistor technology. We have examined all of the currently explored options and found that none of these options are suitable as silicon IC's replacements. In this paper we provide fundamental device criteria that must be satisfied for the successful operation of a manufacturable, not yet invented, device. The two fundamental limits are the removal of heat and reliability. The switching speed of any practical man-made computing device will be in the range of 10(-15) to 10(-3) s. Heisenberg's uncertainty principle and the computer architecture set the heat generation limit. The thermal conductivity of the materials used in the fabrication of a nanodimensional device sets the heat removal limit. In current electronic products, redundancy plays a significant part in improving the reliability of parts with macroscopic defects. In the future, microscopic and even nanoscopic defects will play a critical role in the reliability of disruptive nanoelectronics. The lattice vibrations will set the intrinsic reliability of future computing systems. The two critical limits discussed in this paper provide criteria for the selection of materials used in the fabrication of future devices. Our work shows that diamond contains the clue to providing computing devices that will surpass the performance of silicon-based nanoelectronics.

  5. Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones

    Directory of Open Access Journals (Sweden)

    Morioka Mizue

    2003-08-01

    Full Text Available Abstract Background It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents. Results We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX, a fluoroquinolone (FQ, induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs. Conclusion The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

  6. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9.

    Science.gov (United States)

    Chen, Yongchang; Zheng, Yinghui; Kang, Yu; Yang, Weili; Niu, Yuyu; Guo, Xiangyu; Tu, Zhuchi; Si, Chenyang; Wang, Hong; Xing, Ruxiao; Pu, Xiuqiong; Yang, Shang-Hsun; Li, Shihua; Ji, Weizhi; Li, Xiao-Jiang

    2015-07-01

    CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Endocrine-disrupting chemicals and obesity development in humans: A review

    DEFF Research Database (Denmark)

    Tang-Péronard, Jeanett; Andersen, Helle Raun; Jensen, Tina Kold

    2011-01-01

    This study reviewed the literature on the relations between exposure to chemicals with endocrine-disrupting abilities and obesity in humans. The studies generally indicated that exposure to some of the endocrine-disrupting chemicals was associated with an increase in body size in humans. The resu...... predisposing to later weight gain. The study findings suggest that some endocrine disruptors may play a role for the development of the obesity epidemic, in addition to the more commonly perceived putative contributors.......This study reviewed the literature on the relations between exposure to chemicals with endocrine-disrupting abilities and obesity in humans. The studies generally indicated that exposure to some of the endocrine-disrupting chemicals was associated with an increase in body size in humans...

  8. Regulation of mTOR activity in Snell dwarf and GH receptor gene-disrupted mice.

    Science.gov (United States)

    Dominick, Graham; Berryman, Darlene E; List, Edward O; Kopchick, John J; Li, Xinna; Miller, Richard A; Garcia, Gonzalo G

    2015-02-01

    The involvement of mammalian target of rapamycin (mTOR) in lifespan control in invertebrates, calorie-restricted rodents, and extension of mouse lifespan by rapamycin have prompted speculation that diminished mTOR function may contribute to mammalian longevity in several settings. We show here that mTOR complex-1 (mTORC1) activity is indeed lower in liver, muscle, heart, and kidney tissue of Snell dwarf and global GH receptor (GHR) gene-disrupted mice (GHR-/-), consistent with previous studies. Surprisingly, activity of mTORC2 is higher in fasted Snell and GHR-/- than in littermate controls in all 4 tissues tested. Resupply of food enhanced mTORC1 activity in both controls and long-lived mutant mice but diminished mTORC2 activity only in the long-lived mice. Mice in which GHR has been disrupted only in the liver do not show extended lifespan and also fail to show the decline in mTORC1 and increase in mTORC2 seen in mice with global loss of GHR. The data suggest that the antiaging effects in the Snell dwarf and GHR-/- mice are accompanied by both a decline in mTORC1 in multiple organs and an increase in fasting levels of mTORC2. Neither the lifespan nor mTOR effects appear to be mediated by direct GH effects on liver or by the decline in plasma IGF-I, a shared trait in both global and liver-specific GHR-/- mice. Our data suggest that a more complex pattern of hormonal effects and intertissue interactions may be responsible for regulating both lifespan and mTORC2 function in these mouse models of delayed aging.

  9. Rare copy number variants observed in hereditary breast cancer cases disrupt genes in estrogen signaling and TP53 tumor suppression network.

    Directory of Open Access Journals (Sweden)

    Katri Pylkäs

    Full Text Available Breast cancer is the most common cancer in women in developed countries, and the contribution of genetic susceptibility to breast cancer development has been well-recognized. However, a great proportion of these hereditary predisposing factors still remain unidentified. To examine the contribution of rare copy number variants (CNVs in breast cancer predisposition, high-resolution genome-wide scans were performed on genomic DNA of 103 BRCA1, BRCA2, and PALB2 mutation negative familial breast cancer cases and 128 geographically matched healthy female controls; for replication an independent cohort of 75 similarly mutation negative young breast cancer patients was used. All observed rare variants were confirmed by independent methods. The studied breast cancer cases showed a consistent increase in the frequency of rare CNVs when compared to controls. Furthermore, the biological networks of the disrupted genes differed between the two groups. In familial cases the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity, including DNA double-strand break repair (P = 0.0211. Biological network analysis in the two independent breast cancer cohorts showed that the disrupted genes were closely related to estrogen signaling and TP53 centered tumor suppressor network. These results suggest that rare CNVs represent an alternative source of genetic variation influencing hereditary risk for breast cancer.

  10. Rare copy number variants observed in hereditary breast cancer cases disrupt genes in estrogen signaling and TP53 tumor suppression network.

    Science.gov (United States)

    Pylkäs, Katri; Vuorela, Mikko; Otsukka, Meeri; Kallioniemi, Anne; Jukkola-Vuorinen, Arja; Winqvist, Robert

    2012-01-01

    Breast cancer is the most common cancer in women in developed countries, and the contribution of genetic susceptibility to breast cancer development has been well-recognized. However, a great proportion of these hereditary predisposing factors still remain unidentified. To examine the contribution of rare copy number variants (CNVs) in breast cancer predisposition, high-resolution genome-wide scans were performed on genomic DNA of 103 BRCA1, BRCA2, and PALB2 mutation negative familial breast cancer cases and 128 geographically matched healthy female controls; for replication an independent cohort of 75 similarly mutation negative young breast cancer patients was used. All observed rare variants were confirmed by independent methods. The studied breast cancer cases showed a consistent increase in the frequency of rare CNVs when compared to controls. Furthermore, the biological networks of the disrupted genes differed between the two groups. In familial cases the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity, including DNA double-strand break repair (P = 0.0211). Biological network analysis in the two independent breast cancer cohorts showed that the disrupted genes were closely related to estrogen signaling and TP53 centered tumor suppressor network. These results suggest that rare CNVs represent an alternative source of genetic variation influencing hereditary risk for breast cancer.

  11. Interactive Effects of Prenatal Antidepressant Exposure and Likely Gene Disrupting Mutations on the Severity of Autism Spectrum Disorder

    Science.gov (United States)

    Ackerman, Sean; Schoenbrun, Sarah; Hudac, Caitlin; Bernier, Raphael

    2017-01-01

    To examine the interactive effects of two proposed risk factors which may contribute to symptom severity of Autism Spectrum Disorder (ASD): prenatal antidepressant exposure and likely gene-disrupting (LGD) mutations. Participants included 2748 individuals with ASD from the Simons Simplex Collection. We examined the effects of prenatal…

  12. Improvement of acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae by disruption of the FPS1 aquaglyceroporin gene.

    Science.gov (United States)

    Zhang, Jun-Guo; Liu, Xiu-Ying; He, Xiu-Ping; Guo, Xue-Na; Lu, Ying; Zhang, Bo-Run

    2011-02-01

    The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.

  13. The leading-edge: the significance of sentence disruptions in the development of grammar.

    Science.gov (United States)

    Rispoli, M; Hadley, P

    2001-10-01

    This research explored the relationship between sentence disruptions and the length and complexity of sentences spoken by children developing grammar. The study was cross-sectional in design and used samples of naturalistic, conversational interaction between 26 typically developing children (ages 2;6 to 4;0) and a primary caregiver. The active, declarative sentences produced by these children were coded for the presence of disruption, length in morphemes and words, and clausal complexity. The results showed that, for the majority of the children, disrupted sentences tended to be longer and more complex than fluent sentences. The magnitude of the differences in length and complexity was positively correlated with the children's grammatical development, as measured by the Index of Productive Syntax. It was also found that differences between the average complexity of disrupted versus fluent sentences increased with grammatical development even when sentence length was held constant. As grammatical development proceeded, disrupted sentences were more apt to be sentences on the "leading-edge" of the child's production capacity. Although these more advanced grammatical structures are part of the child's grammatical competence, the child cannot produce these sentences without an increased risk of processing difficulty. The results are congruent with proposals concerning the incremental and procedural nature of adult sentence production.

  14. Gene Expression Profiling Identifies Lobe-Specific and Common Disruptions of Multiple Gene Networks in Testosterone-Supported, 17β-Estradiol- or Diethylstilbestrol-Induced Prostate Dysplasia in Noble Rats

    Directory of Open Access Journals (Sweden)

    Neville N.C. Tam

    2008-01-01

    Full Text Available The xenoestrogen diethylstilbestrol (DES is commonly believed to mimic the action of the natural estrogen 17β-estradiol (E2. To determine if these two estrogens exert similar actions in prostate carcinogenesis, we elevated circulating levels of estrogen in Noble (NBL rats with E2/DES-filled implants, while maintaining physiological levels of testosterone (T in the animals with T-filled implants. The two estrogens induced dysplasia in a lobe-specific manner, with E2 targeting only the lateral prostate (LP and DES impacting only the ventral prostate (VP. Gene expression profiling identified distinct and common E2-disrupted versus DES-disrupted gene networks in each lobe. More importantly, hierarchical clustering analyses revealed that T + E2 treatment primarily affected the gene expression pattern in the LP, whereas T + DES treatment primarily affected the gene expression profile in the VP. Gene ontology analyses and pathway mapping suggest that the two hormone treatments disrupt unique and/or common cellular processes, including cell development, proliferation, motility, apoptosis, and estrogen signaling, which may be linked to dysplasia development in the rat prostate. These findings suggest that the effects of xenoestrogens and natural estrogens on the rat prostate are more divergent than previously suspected and that these differences may explain the lobe-specific carcinogenic actions of the hormones.

  15. Targeted p120-catenin ablation disrupts dental enamel development

    DEFF Research Database (Denmark)

    Bartlett, John D; Dobeck, Justine M; Tye, Coralee E

    2010-01-01

    Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by...... to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.......Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide...... by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate...

  16. Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

    Science.gov (United States)

    Bartlett, John D.; Dobeck, Justine M.; Tye, Coralee E.; Perez-Moreno, Mirna; Stokes, Nicole; Reynolds, Albert B.; Fuchs, Elaine; Skobe, Ziedonis

    2010-01-01

    Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows. PMID:20862276

  17. Targeted p120-catenin ablation disrupts dental enamel development.

    Directory of Open Access Journals (Sweden)

    John D Bartlett

    2010-09-01

    Full Text Available Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ, and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.

  18. Disruptive and Sustaining Technology Development Approaches in Defense Acquisition

    Science.gov (United States)

    2014-04-30

    from the market in developing technology solutions is a viable strategy in a competitive market (Lampel & Shamsie, 2003; Mintzberg , 1979; Mintzberg ...and process. Academy of Management Review, 3(3), 546-562. Mintzberg , H. (1979). Patterns in strategy formation. International Studies of...Management & Organization, 9(3), 67-86. Mintzberg , H., & McHugh, A. (1985). Strategy formation in an adhocracy. Administrative Science Quarterly, 30(2

  19. Gene network analysis of Arabidopsis thaliana flower development through dynamic gene perturbations.

    Science.gov (United States)

    Ó'Maoiléidigh, Diarmuid S; Thomson, Bennett; Raganelli, Andrea; Wuest, Samuel E; Ryan, Patrick T; Kwaśniewska, Kamila; Carles, Cristel C; Graciet, Emmanuelle; Wellmer, Frank

    2015-07-01

    Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  20. Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

    Directory of Open Access Journals (Sweden)

    Meijia Yang

    2017-09-01

    Full Text Available BRAF-V600E (1799T > A is one of the most frequently reported driver mutations in multiple types of cancers, and patients with such mutations could benefit from selectively inactivating the mutant allele. Near this mutation site, there are two TTTN and one NGG protospacer-adjacent motifs (PAMs for Cpf1 and Cas9 CRISPR nucleases, respectively. The 1799T > A substitution also leads to the occurrence of a novel NGNG PAM for the EQR variant of Cas9. We examined the editing efficacy and selectivity of Cpf1, Cas9, and EQR variant to this mutation site. Only Cpf1 demonstrated robust activity to induce specific disruption of only mutant BRAF, not wild-type sequence. Cas9 recognized and cut both normal and mutant alleles, and no obvious gene editing events were observed using EQR variant. Our results support the potential applicability of Cpf1 in precision medicine through highly specific inactivation of many other gain-of-function mutations.

  1. Water deficit disrupts male gametophyte development in Quercus ilex.

    Science.gov (United States)

    Bykova, Olga; Limousin, Jean-Marc; Ourcival, Jean-Marc; Chuine, Isabelle

    2018-01-19

    Tree species distribution, and hence forest biodiversity, rely on the reproductive capacity of trees which is currently affected by climate change. Drought-induced pollen sterility could increase as a consequence of more intense and more frequent droughts projected for temperate and Mediterranean regions and threaten the sexual regeneration of trees in these regions. To evaluate this possibility, we examined the effect of long-term partial rainfall exclusion (-27% of precipitation) on male reproductive development in holm oak, Quercus ilex, one of the most important and widespread tree species of the Mediterranean region. We examined anther area, pollen production, pollen abortion as well as viable pollen production in control and dry treatments. Microscopic examinations revealed significant differences in pollen development between trees in the dry and the control treatments, even though anthesis occurred before the annual drought onset. Our results demonstrate that anthers collected from Q. ilex trees in the dry treatment, which experienced long-term increased drought stress especially during the summer time were the same size as anthers in the control treatment, but displayed 25% pollen abortion and almost 20% reduction in pollen production. Subsequently, the number of viable pollen grains in anthers from dry treatment was 35% less than in control. These results suggest a carry-over effect of drought stress on pollen production that could reduce the reproductive success of Q. ilex. The results of this study have broad implications for better understanding of the determinants of tree reproduction by masting and anticipate the outcomes of expected drought increase in the Mediterranean on forest dynamics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. Chronic Ethanol Consumption Disrupts the Core Molecular Clock and Diurnal Rhythms of Metabolic Genes in the Liver without Affecting the Suprachiasmatic Nucleus

    Science.gov (United States)

    Filiano, Ashley N.; Millender-Swain, Telisha; Johnson, Russell; Young, Martin E.; Gamble, Karen L.; Bailey, Shannon M.

    2013-01-01

    Chronic ethanol consumption disrupts several metabolic pathways including β-oxidation and lipid biosynthesis, facilitating the development of alcoholic fatty liver disease. Many of these same metabolic pathways are directly regulated by cell autonomous circadian clocks, and recent studies suggest that disruption of daily rhythms in metabolism contributes to multiple common cardiometabolic diseases (including non-alcoholic fatty liver disease). However, it is not known whether ethanol disrupts the core molecular clock in the liver, nor whether this, in turn, alters rhythms in lipid metabolism. Herein, we tested the hypothesis that chronic ethanol consumption disrupts the molecular circadian clock in the liver and potentially changes the diurnal expression patterns of lipid metabolism genes. Consistent with previous studies, male C57BL/6J mice fed an ethanol-containing diet exhibited higher levels of liver triglycerides compared to control mice, indicating hepatic steatosis. Further, the diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erbα, and Tef) were altered in livers from ethanol-fed mice. In contrast, ethanol had only minor effects on the expression of core clock genes in the suprachiasmatic nucleus (SCN). These results were confirmed in Per2Luciferase knock-in mice, in which ethanol induced a phase advance in PER2::LUC bioluminescence oscillations in liver, but not SCN. Further, there was greater variability in the phase of PER2::LUC oscillations in livers from ethanol-fed mice. Ethanol consumption also affected the diurnal oscillations of metabolic genes, including Adh1, Cpt1a, Cyp2e1, Pck1, Pdk4, Ppargc1a, Ppargc1b and Srebp1c, in the livers of C57BL/6J mice. In summary, chronic ethanol consumption alters the function of the circadian clock in liver. Importantly, these results suggest that chronic ethanol consumption, at levels sufficient to cause steatosis

  3. Disruption of germination and seedling development in Brassica napus by mutations causing severe seed hormonal imbalance

    Directory of Open Access Journals (Sweden)

    Tung eNguyen

    2016-03-01

    Full Text Available The Brassica napus (oilseed rape accession 1012-98 shows a disturbed germination phenotype that was thought to be associated with its lack of testa pigmentation and thin seed coat. Here we demonstrate that the disturbed germination and seedling development are actually due to independent mutations that disrupt the balance of hormone metabolites and their regulators in the seeds. High-throughput UPLC-MS/MS hormone profiling of seeds and seedlings before and after germination revealed that 1012-98 has a severely disturbed hormone balance with extremely atypical, excessive quantities of auxin and ABA metabolites. The resulting hypersensitivity to abscisic acid (ABA and a corresponding increase in dormancy often results in death of the embryo after imbibition or high frequencies of disturbed, often lethal developmental phenotypes, resembling Arabidopsis mutants for the auxin regulatory factor gene ARF10 or the auxin-overproducing transgenic line iaaM-OX. Molecular cloning of Brassica ARF10 orthologues revealed four loci in normal B. napus, two derived from the Brassica A genome and two from the C genome. On the other hand, the phenotypic mutant 1012-98 exhibited amplification of C-genome BnaC.ARF10 copy number along with a chimeric allele originating from recombination between homoeologous A and C genome loci which lead to minor increase of Bna.ARF10 transcription on the critical timepoint for seed germination, the indirect regulator of ABI3, the germinative inhibitor. Bna.GH3.5 expression was upregulated to conjugate free auxin to IAA-asp between 2-6 DAS. Functional amino acid changes were also found in important DNA binding domains of one BnaC.ARF10 locus, suggesting that regulatory changes in Bna.ARF10 are collectively responsible for the observed phenotpyes in 1012-98. To our knowledge, this study is the first to report disruption of germination and seedling development in Brassica napus caused by the crosstalk of auxin-ABA and the

  4. Disruption of Germination and Seedling Development in Brassica napus by Mutations Causing Severe Seed Hormonal Imbalance.

    Science.gov (United States)

    Nguyen, Tung C T; Obermeier, Christian; Friedt, Wolfgang; Abrams, Suzanne R; Snowdon, Rod J

    2016-01-01

    The Brassica napus (oilseed rape) accession 1012-98 shows a disturbed germination phenotype that was thought to be associated with its lack of testa pigmentation and thin seed coat. Here, we demonstrate that the disturbed germination and seedling development are actually due to independent mutations that disrupt the balance of hormone metabolites and their regulators in the seeds. High-throughput UPLC-MS/MS hormone profiling of seeds and seedlings before and after germination revealed that 1012-98 has a severely disturbed hormone balance with extremely atypical, excessive quantities of auxin and ABA metabolites. The resulting hypersensitivity to abscisic acid (ABA) and a corresponding increase in dormancy often results in death of the embryo after imbibition or high frequencies of disturbed, often lethal developmental phenotypes, resembling Arabidopsis mutants for the auxin regulatory factor gene ARF10 or the auxin-overproducing transgenic line iaaM-OX. Molecular cloning of Brassica ARF10 orthologs revealed four loci in normal B. napus, two derived from the Brassica A genome and two from the C genome. On the other hand, the phenotypic mutant 1012-98 exhibited amplification of C-genome BnaC.ARF10 copy number along with a chimeric allele originating from recombination between homeologous A and C genome loci which lead to minor increase of Bna.ARF10 transcription on the critical timepoint for seed germination, the indirect regulator of ABI3, the germinative inhibitor. Bna.GH3.5 expression was upregulated to conjugate free auxin to IAA-asp between 2 and 6 DAS. Functional amino acid changes were also found in important DNA binding domains of one BnaC.ARF10 locus, suggesting that regulatory changes in Bna.ARF10 are collectively responsible for the observed phenotpyes in 1012-98. To our knowledge, this study is the first to report disruption of germination and seedling development in Brassica napus caused by the crosstalk of auxin-ABA and the corresponding regulators Bna

  5. Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells

    Directory of Open Access Journals (Sweden)

    David L DiGiusto

    2016-01-01

    Full Text Available Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5 is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC. The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov.

  6. A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development.

    Directory of Open Access Journals (Sweden)

    Konstantinos Zarbalis

    2004-08-01

    Full Text Available Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice (Boncinelli et al. 2000; Molnar and Hannan 2000; Walsh and Goffinet 2000, many questions remain unanswered. To provide resources for further dissecting cortical development, we have carried out a focused screen for recessive mutations that disrupt cortical development. One aim of the screen was to identify mutants that disrupt the tangential migration of interneurons into the cortex. At the same time, we also screened for mutations that altered the growth or morphology of the cerebral cortex. We report here the identification of thirteen mutants with defects in aspects of cortical development ranging from the establishment of epithelial polarity to the invasion of thalamocortical axons. Among the collection are three novel alleles of genes for which mutant alleles had already been used to explore forebrain development, and four mutants with defects in interneuron migration. The mutants that we describe here will aid in deciphering the molecules and mechanisms that regulate cortical development. Our results also highlight the utility of focused screens in the mouse, in addition to the large-scale and broadly targeted screens that are being carried out at mutagenesis centers.

  7. Improved production of enzymes, which are expressed under the Pho regulon promoter, in the rmf gene (encoding ribosome modulation factor) disruptant of Escherichia coli.

    Science.gov (United States)

    Imaizumi, Akira; Koseki, Chie; Matsui, Kazuhiko; Kojima, Hiroyuki

    2006-04-01

    Using a DNA macroarray, we investigated the effects of rmf gene (encoding ribosome modulation factor) disruption on gene expression profiles in Escherichia coli. This strain showed a phosphate-starvation-like response in gene expression even under phosphate sufficient conditions; significant upregulation of the Pho regulon genes was observed. Further, the production of alkaline phosphatase, a product of the Pho regulon gene, phoA, increased in the rmf disruptant under a Pi sufficient condition. Furthermore, production of PhoC acid phosphatase/nucleoside pyrophosphate phosphotransferase derived from Morganella morganii also increased significantly in the rmf disruptant. We concluded that host modification by the rmf gene disruption has potential benefit in industrial enzyme production using Escherichia coli.

  8. Is the Mobile Phone a Disruptive Technology? A Partial Review of Evidence from Developing Countries

    NARCIS (Netherlands)

    Columbus, Simon

    2012-01-01

    The authors of this chapter provide an inter-disciplinary review of studies on economic impacts of mobile telephony in developing countries, giving particular attention to the disruptive potential of the technology and its associated social practices. Four major areas of impact are identified: the

  9. Development and validation of in vitro bioassays for thyroid hormone receptor mediated endocrine disruption

    NARCIS (Netherlands)

    Freitas, de J.

    2012-01-01

    Thyroid hormones regulate crucial processes in vertebrates such as reproduction, development and energy metabolism. Endocrine disruption via the thyroid hormone system is gaining more attention both from scientists and regulators, because of the increasing incidence of hormone-related cancers and

  10. Detecting the effects of environmentally relevant concentrations of thyroid hormone disrupting compounds on amphibian development

    NARCIS (Netherlands)

    Gutleb, A.C.

    2006-01-01

    Persistent organic pollutants such as PCBs have been hypothesized to contribute to the observed global decline of amphibian populations. Thyroid hormone (TH) disruption is one of the possible mechanisms for effects of xenobiotics on amphibian development. In addition to the important functions

  11. The Need for Mobile Application Development in IS Curricula: An Innovation and Disruptive Technologies Perspective

    Science.gov (United States)

    Babb, Jeffry S., Jr.; Abdullat, Amjad

    2012-01-01

    Disruptive technologies, such as mobile applications development, will always present a dilemma for Information Systems educators as dominant paradigms in our environment will tend to favor the existing sustaining technologies that we have become known for in our discipline. In light of this friction, we share our approach in investigating and…

  12. Mixtures of environmentally relevant endocrine disrupting chemicals affect mammary gland development in female and male rats

    DEFF Research Database (Denmark)

    Mandrup, Karen Riiber; Johansson, Hanna Katarina Lilith; Boberg, Julie

    2015-01-01

    Estrogenic chemicals are able to alter mammary gland development in female rodents, but little is known on the effects of anti-androgens and mixtures of endocrine disrupting chemicals (EDCs) with dissimilar modes of action. Pregnant rat dams were exposed during gestation and lactation to mixtures...

  13. Bacterial niche-specific genome expansion is coupled with highly frequent gene disruptions in deep-sea sediments

    KAUST Repository

    Wang, Yong

    2011-12-21

    The complexity and dynamics of microbial metagenomes may be evaluated by genome size, gene duplication and the disruption rate between lineages. In this study, we pyrosequenced the metagenomes of microbes obtained from the brine and sediment of a deep-sea brine pool in the Red Sea to explore the possible genomic adaptations of the microbes in response to environmental changes. The microbes from the brine and sediments (both surface and deep layers) of the Atlantis II Deep brine pool had similar communities whereas the effective genome size varied from 7.4 Mb in the brine to more than 9 Mb in the sediment. This genome expansion in the sediment samples was due to gene duplication as evidenced by enrichment of the homologs. The duplicated genes were highly disrupted, on average by 47.6% and 70% for the surface and deep layers of the Atlantis II Deep sediment samples, respectively. The disruptive effects appeared to be mainly due to point mutations and frameshifts. In contrast, the homologs from the Atlantis II Deep brine sample were highly conserved and they maintained relatively small copy numbers. Likely, the adaptation of the microbes in the sediments was coupled with pseudogenizations and possibly functional diversifications of the paralogs in the expanded genomes. The maintenance of the pseudogenes in the large genomes is discussed. © 2011 Wang et al.

  14. Bacterial niche-specific genome expansion is coupled with highly frequent gene disruptions in deep-sea sediments.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available The complexity and dynamics of microbial metagenomes may be evaluated by genome size, gene duplication and the disruption rate between lineages. In this study, we pyrosequenced the metagenomes of microbes obtained from the brine and sediment of a deep-sea brine pool in the Red Sea to explore the possible genomic adaptations of the microbes in response to environmental changes. The microbes from the brine and sediments (both surface and deep layers of the Atlantis II Deep brine pool had similar communities whereas the effective genome size varied from 7.4 Mb in the brine to more than 9 Mb in the sediment. This genome expansion in the sediment samples was due to gene duplication as evidenced by enrichment of the homologs. The duplicated genes were highly disrupted, on average by 47.6% and 70% for the surface and deep layers of the Atlantis II Deep sediment samples, respectively. The disruptive effects appeared to be mainly due to point mutations and frameshifts. In contrast, the homologs from the Atlantis II Deep brine sample were highly conserved and they maintained relatively small copy numbers. Likely, the adaptation of the microbes in the sediments was coupled with pseudogenizations and possibly functional diversifications of the paralogs in the expanded genomes. The maintenance of the pseudogenes in the large genomes is discussed.

  15. Disrupted Lung Development and Bronchopulmonary Dysplasia: Opportunities for Lung Repair and Regeneration

    Science.gov (United States)

    Baker, Christopher D.; Alvira, Cristina M.

    2014-01-01

    Purpose of review Advances of medical therapy have increased survival of extremely premature infants, and changed the pathology of bronchopulmonary dysplasia (BPD) from one of acute lung injury to a disease of disrupted lung development. With this evolution, new questions emerge regarding: (i) the molecular mechanisms that control postnatal lung development; (ii) the effect of early disruptions of postnatal lung development on long-term lung function; and (iii) the existence of endogenous mechanisms that permit lung regeneration after injury. Recent Findings Recent data demonstrate that a significant component of alveolarization, the final stage of lung development, occurs postnatally. Further, clinical and experimental studies demonstrate that premature birth disrupts alveolarization, decreasing the gas exchange surface area of the lung and causing BPD. BPD is associated with significant short-term morbidity, and new longitudinal, clinical data demonstrate that survivors of BPD have long-standing deficits in lung function, and may be at risk for the development of additional lung disease as adults. Unfortunately, current care is mainly supportive with few effective therapies that prevent or treat established BPD. These studies underscore the need to further elucidate the mechanisms that direct postnatal lung growth, and develop innovative strategies to stimulate lung regeneration. Summary Despite significant improvements in the care and survival of extremely premature infants, BPD remains a major clinical problem. While efforts should remain focused on the prevention of preterm labor and BPD, novel research aimed at promoting postnatal alveolarization offers a unique opportunity to develop effective strategies to treat established BPD. PMID:24739494

  16. Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6).

    Science.gov (United States)

    Morcx, Serena; Kunz, Caroline; Choquer, Mathias; Assie, Sébastien; Blondet, Eddy; Simond-Côte, Elisabeth; Gajek, Karina; Chapeland-Leclerc, Florence; Expert, Dominique; Soulie, Marie-Christine

    2013-03-01

    Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. A functional polymorphism in the disrupted-in schizophrenia 1 gene is associated with chronic fatigue syndrome.

    Science.gov (United States)

    Fukuda, Sanae; Hashimoto, Ryota; Ohi, Kazutaka; Yamaguti, Kouzi; Nakatomi, Yasuhito; Yasuda, Yuka; Kamino, Kouzin; Takeda, Masatoshi; Tajima, Seiki; Kuratsune, Hirohiko; Nishizawa, Yoshiki; Watanabe, Yasuyoshi

    2010-05-08

    Disrupted-in schizophrenia 1 (DISC1), identified in a pedigree with a familial psychosis with the chromosome translocation (1:11), is a putative susceptibility gene for psychoses such as schizophrenia and major depressive disorder (MDD). Patients with chronic fatigue syndrome (CFS) report having continuous severe fatigue and many overlapping symptoms with MDD; however, the mechanism and effective treatment of CFS are still unclear. We focused on the overlapping symptoms between CFS and MDD and performed an association study of the functional single-nucleotide polymorphism (SNP) in the DISC1 gene with CFS. Venous blood was drawn from CFS patients and controls and genomic DNA was extracted from the whole blood according to standard procedures. Ser704Cys DISC1 SNP was genotyped using the TaqMan 5'-exonuclease allelic discrimination assay. We found that the Cys704 allele of Ser704Cys SNP was associated with an increased risk of CFS development compared with the Ser704 allele. DISC1 Ser704Cys might be a functional variant that affects one of the mechanisms implicated in the biology of CFS. Some patients with CFS showed a phenotype similar to that of patients with MDD, but further studies are needed to clarify the biological mechanism, because this study is of a rather preliminary nature. Despite the variety of patients with CFS, DISC1 Ser704Cys has an association with CFS, which may also suggest that DISC1 plays a central role in the induction of various psychiatric diseases. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Circadian clock genes Per1 and Per2 regulate the response of metabolism-associated transcripts to sleep disruption.

    Directory of Open Access Journals (Sweden)

    Jana Husse

    Full Text Available Human and animal studies demonstrate that short sleep or poor sleep quality, e.g. in night shift workers, promote the development of obesity and diabetes. Effects of sleep disruption on glucose homeostasis and liver physiology are well documented. However, changes in adipokine levels after sleep disruption suggest that adipocytes might be another important peripheral target of sleep. Circadian clocks regulate metabolic homeostasis and clock disruption can result in obesity and the metabolic syndrome. The finding that sleep and clock disruption have very similar metabolic effects prompted us to ask whether the circadian clock machinery may mediate the metabolic consequences of sleep disruption. To test this we analyzed energy homeostasis and adipocyte transcriptome regulation in a mouse model of shift work, in which we prevented mice from sleeping during the first six hours of their normal inactive phase for five consecutive days (timed sleep restriction--TSR. We compared the effects of TSR between wild-type and Per1/2 double mutant mice with the prediction that the absence of a circadian clock in Per1/2 mutants would result in a blunted metabolic response to TSR. In wild-types, TSR induces significant transcriptional reprogramming of white adipose tissue, suggestive of increased lipogenesis, together with increased secretion of the adipokine leptin and increased food intake, hallmarks of obesity and associated leptin resistance. Some of these changes persist for at least one week after the end of TSR, indicating that even short episodes of sleep disruption can induce prolonged physiological impairments. In contrast, Per1/2 deficient mice show blunted effects of TSR on food intake, leptin levels and adipose transcription. We conclude that the absence of a functional clock in Per1/2 double mutants protects these mice from TSR-induced metabolic reprogramming, suggesting a role of the circadian timing system in regulating the physiological effects

  19. Development and validation of the Johns Hopkins Disruptive Clinician Behavior Survey.

    Science.gov (United States)

    Dang, Deborah; Nyberg, Dorothy; Walrath, Jo M; Kim, Miyong T

    2015-01-01

    Although the negative impact of disruptive clinician behavior on quality health care delivery has gained attention recently, little systematic effort to address this issue has been reported. To facilitate empirical research to reduce disruptive clinician behaviors, an assessment tool (Johns Hopkins Disruptive Clinician Behavior Survey [JH-DCBS]) with 5 discrete subscales was developed using a 2-step design. First a pool of items was generated from focus group studies and the literature, and then a psychometric evaluation of the survey was conducted with a sample of clinicians (N = 1198) practicing in a large urban academic medical center. The results indicated that the tool was reliable (Cronbach α = .79-.91), showed high content validity (Content Validity Index = .97), and had significantly high correlations with theoretically selected variables. The study team concluded that the JH-DCBS provides a valid empirical assessment of disruptive behavior. Assessment results may be used to design strategies to improve the health and safety of practice environments. © The Author(s) 2014.

  20. Genetic programs of the developing tuberal hypothalamus and potential mechanisms of their disruption by environmental factors.

    Science.gov (United States)

    Nesan, Dinushan; Kurrasch, Deborah M

    2016-12-15

    The hypothalamus is a critical regulator of body homeostasis, influencing the autonomic nervous system and releasing trophic hormones to modulate the endocrine system. The developmental mechanisms that govern formation of the mature hypothalamus are becoming increasingly understood as research in this area grows, leading us to gain appreciation for how these developmental programs are susceptible to disruption by maternal exposure to endocrine disrupting chemicals or other environmental factors in utero. These vulnerabilities, combined with the prominent roles of the various hypothalamic nuclei in regulating appetite, reproductive behaviour, mood, and other physiologies, create a window whereby early developmental disruption can have potent long-term effects. Here we broadly outline our current understanding of hypothalamic development, with a particular focus on the tuberal hypothalamus, including what is know about nuclear coalescing and maturation. We finish by discussing how exposure to environmental or maternally-derived factors can perhaps disrupt these hypothalamic developmental programs, and potentially lead to neuroendocrine disease states. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Effects of Exposure to the Endocrine-Disrupting Chemical Bisphenol A During Critical Windows of Murine Pituitary Development.

    Science.gov (United States)

    Eckstrum, Kirsten S; Edwards, Whitney; Banerjee, Annesha; Wang, Wei; Flaws, Jodi A; Katzenellenbogen, John A; Kim, Sung Hoon; Raetzman, Lori T

    2018-01-01

    Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 μg/kg/d BPA, environmentally relevant doses, or 50 μg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 μg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 μg/kg/d BPA in males and at 0.5 and 50 μg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-β in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms. Copyright © 2018 Endocrine Society.

  2. Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption.

    Science.gov (United States)

    Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

    2017-10-27

    CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo. © 2017 American Heart Association, Inc.

  3. GAP1, a novel selection and counter-selection marker for multiple gene disruptions in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Hansen, J.

    2000-01-01

    We report on the use of a new homologous marker for use in multiple gene deletions in S, cerevisiae, the general amino acid permease gene (GAP1), A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine, L-citrulline as......We report on the use of a new homologous marker for use in multiple gene deletions in S, cerevisiae, the general amino acid permease gene (GAP1), A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine, L...... flanked by short (60 bp) stretches of the gene in question. Through homologous recombination, the cassette will integrate into the target gene, which is thus replaced by GAP1, and mutants are selected for on minimal L-citrulline medium. When propagated under non-selective conditions, some cells will lose...... the GAP1 gene. This is caused by recombination between two Salmonella typuimurium hisG direct repeats embracing GAP1, and will result in a sub-population of gap1 cells. Such cells are selected on a medium containing D-histidine, and may subsequently be used for a second gene disruption. Hence, multiple...

  4. Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2007-01-01

    Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

  5. Disruption of the acyl-coa binding protein gene delays hepatic adaptation to metabolic changes at weaning

    DEFF Research Database (Denmark)

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek

    2011-01-01

    , little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice go through a crisis with overall weakness, and a slightly decreased growth...

  6. Disruption of Wnt/β-catenin signaling in odontoblasts and cementoblasts arrests tooth root development in postnatal mouse teeth.

    Science.gov (United States)

    Zhang, Ran; Yang, Guan; Wu, Ximei; Xie, Jing; Yang, Xiao; Li, Tiejun

    2013-01-01

    Tooth development undergoes a series of complex reciprocal interactions between dental epithelium and the underlying mesenchymal cells. Compared with the study in tooth crown formation, little is known about the molecular mechanism underlying the development of tooth roots. In the present study, we conditionally knock out β-catenin gene (Ctnnb1) within developing odontoblasts and cementoblasts during the development of tooth roots, and observed rootless molars as well as incomplete incisors. Histological analyses revealed intact structure of molar crown and labial side of incisor, however, as for the molar roots and the lingual portion of incisor, the formation of dentin and periodontal tissues were greatly hampered. In situ hybridization experiments using probes of odontoblastic marker genes collagen type I, alpha 1 (Col1a1), osteocalcin (OC) and dentin sialophosphoprotein (Dspp) manifested striking undifferentiation of root odontoblasts in which Ctnnb1 was eliminated. Bromodeoxyuridine (BrdU) labeling and proliferating cell nuclear antigen (PCNA) immunohistochemical experiments also showed retarded proliferation of pre-odontoblasts in mutant mice. However, cell apoptosis was not affected. Additionally, a disrupted formation of cementoblasts, suggested by the absence of transcripts of bone sialoprotein (Bsp) in follicle mesenchyme, was also evident in mutant mice. Our study provides strong in vivo evidence to confirm that Wnt/β-catenin signaling is functionally significant to root odontogenesis and cementogenesis during the tooth root development.

  7. Disrupted auto-regulation of the spliceosomal gene SNRPB causes cerebro–costo–mandibular syndrome

    Science.gov (United States)

    Lynch, Danielle C.; Revil, Timothée; Schwartzentruber, Jeremy; Bhoj, Elizabeth J.; Innes, A. Micheil; Lamont, Ryan E.; Lemire, Edmond G.; Chodirker, Bernard N.; Taylor, Juliet P.; Zackai, Elaine H.; McLeod, D. Ross; Kirk, Edwin P.; Hoover-Fong, Julie; Fleming, Leah; Savarirayan, Ravi; Boycott, Kym; MacKenzie, Alex; Brudno, Michael; Bulman, Dennis; Dyment, David; Majewski, Jacek; Jerome-Majewska, Loydie A.; Parboosingh, Jillian S.; Bernier, Francois P.

    2014-01-01

    Elucidating the function of highly conserved regulatory sequences is a significant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebro–costo–mandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development. PMID:25047197

  8. Disrupted auto-regulation of the spliceosomal gene SNRPB causes cerebro-costo-mandibular syndrome.

    Science.gov (United States)

    Lynch, Danielle C; Revil, Timothée; Schwartzentruber, Jeremy; Bhoj, Elizabeth J; Innes, A Micheil; Lamont, Ryan E; Lemire, Edmond G; Chodirker, Bernard N; Taylor, Juliet P; Zackai, Elaine H; McLeod, D Ross; Kirk, Edwin P; Hoover-Fong, Julie; Fleming, Leah; Savarirayan, Ravi; Majewski, Jacek; Jerome-Majewska, Loydie A; Parboosingh, Jillian S; Bernier, Francois P

    2014-07-22

    Elucidating the function of highly conserved regulatory sequences is a significant challenge in genomics today. Certain intragenic highly conserved elements have been associated with regulating levels of core components of the spliceosome and alternative splicing of downstream genes. Here we identify mutations in one such element, a regulatory alternative exon of SNRPB as the cause of cerebro-costo-mandibular syndrome. This exon contains a premature termination codon that triggers nonsense-mediated mRNA decay when included in the transcript. These mutations cause increased inclusion of the alternative exon and decreased overall expression of SNRPB. We provide evidence for the functional importance of this conserved intragenic element in the regulation of alternative splicing and development, and suggest that the evolution of such a regulatory mechanism has contributed to the complexity of mammalian development.

  9. Development and application of QSAR models for mechanisms related to endocrine disruption

    DEFF Research Database (Denmark)

    Abildgaard Rosenberg, Sine

    is a background section, comprising 1) an introduction to the endocrine system with a focus on thyroid hormones (THs) and their essential function in neurodevelopment as well as a description of how chemicals may interference with endocrine mechanisms and cause adverse effects, 2) an introduction to the applied......Humans are daily exposed to a wide variety of man-made chemicals through food, consumer products, water, air inhalation etc. For the main part of these chemicals no or only very limited information is available on their potential to cause endocrine disruption. Traditionally such information has...... information on the mode of action of chemicals in a faster and cheaper way. The main purpose in this PhD project was to develop QSAR models for mechanisms related to endocrine disruption and apply the models to predict 10,000s of chemicals to which humans are potentially exposed. The first part of the thesis...

  10. Synergistic Disruption of External Male Sex Organ Development by a Mixture of Four Antiandrogens

    DEFF Research Database (Denmark)

    Christiansen, Sofie; Scholze, Martin; Dalgaard, Majken

    2009-01-01

    By disrupting the action of androgens during gestation, certain chemicals present in food, consumer products and the environment can induce irreversible demasculinisation and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals...... are not well described, especially when they exert their actions by differing molecular mechanisms. Objectives: To fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP), two fungicides present in food, vinclozolin and prochloraz......, and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance, retained nipples, sex organ weights and malformations of genitalia. These chemicals were chosen because they disrupt androgen action according to differing mechanisms of action. Results...

  11. Disruption of phenylalanine hydroxylase reduces adult lifespan and fecundity, and impairs embryonic development in parthenogenetic pea aphids.

    Science.gov (United States)

    Simonet, Pierre; Gaget, Karen; Parisot, Nicolas; Duport, Gabrielle; Rey, Marjolaine; Febvay, Gérard; Charles, Hubert; Callaerts, Patrick; Colella, Stefano; Calevro, Federica

    2016-10-03

    Phenylalanine hydroxylase (PAH) is a key tyrosine-biosynthetic enzyme involved in neurological and melanin-associated physiological processes. Despite extensive investigations in holometabolous insects, a PAH contribution to insect embryonic development has never been demonstrated. Here, we have characterized, for the first time, the PAH gene in a hemimetabolous insect, the aphid Acyrthosiphon pisum. Phylogenetic and sequence analyses confirmed that ApPAH is closely related to metazoan PAH, exhibiting the typical ACT regulatory and catalytic domains. Temporal expression patterns suggest that ApPAH has an important role in aphid developmental physiology, its mRNA levels peaking at the end of embryonic development. We used parental dsApPAH treatment to generate successful knockdown in aphid embryos and to study its developmental role. ApPAH inactivation shortens the adult aphid lifespan and considerably affects fecundity by diminishing the number of nymphs laid and impairing embryonic development, with newborn nymphs exhibiting severe morphological defects. Using single nymph HPLC analyses, we demonstrated a significant tyrosine deficiency and a consistent accumulation of the upstream tyrosine precursor, phenylalanine, in defective nymphs, thus confirming the RNAi-mediated disruption of PAH activity. This study provides first insights into the role of PAH in hemimetabolous insects and demonstrates that this metabolic gene is essential for insect embryonic development.

  12. Loss of human ribosomal gene CpG methylation enhances cryptic RNA polymerase II transcription and disrupts ribosomal RNA processing.

    Science.gov (United States)

    Gagnon-Kugler, Thérèse; Langlois, Frédéric; Stefanovsky, Victor; Lessard, Frédéric; Moss, Tom

    2009-08-28

    Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to downregulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an explanation for the cytotoxic effects of loss of CpG methylation.

  13. Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets

    National Research Council Canada - National Science Library

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S; Chen, Juan; Zhang, Yulong; Welsh, Michael J; Leno, Gregory H; Engelhardt, John F

    2008-01-01

    .... In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning...

  14. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    Science.gov (United States)

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  15. Growth and development of maize that contains mutant tubulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  16. Maternal exposure to di-(2-ethylhexyl) phthalate disrupts placental growth and development in pregnant mice

    Energy Technology Data Exchange (ETDEWEB)

    Zong, Teng; Lai, Lidan [Department of Physiology, School of Medicine, Nanchang University, Nanchang, Jiangxi 330006 (China); Hu, Jia [Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi (China); Guo, Meijun; Li, Mo; Zhang, Lu; Zhong, Chengxue; Yang, Bei; Wu, Lei; Zhang, Dalei; Tang, Min [Department of Physiology, School of Medicine, Nanchang University, Nanchang, Jiangxi 330006 (China); Kuang, Haibin, E-mail: kuanghaibin@ncu.edu.cn [Department of Physiology, School of Medicine, Nanchang University, Nanchang, Jiangxi 330006 (China)

    2015-10-30

    Highlights: • The influence of DEHP on the development of placenta was investigated. • DEHP disrupts the growth and development of placenta. • DEHP disrupts the formation of labyrinth vascularization. • DEHP inhibits the proliferation of ectoplacental cone and placenta. • DEHP induces the apoptosis of placenta via activated MAPK signaling pathway. - Abstract: Di-(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer and widely dispersed in the environment. DEHP exposure reduces embryo implantations, increases embryonic loss, and decreases fetal body weights. However, no detailed information is available about the effect of DEHP on the placentation during pregnancy. Thus, our aim was to explore the effect of DEHP on the growth and development of placenta in vivo. Mice were administered DEHP by gavages at 125, 250, 500 mg/kg/day from gestational days (GD) 1 until sacrifice. Results showed that DEHP treatment significantly reduced the weight of placenta at GD 13. Histopathologically, in DEHP-treated group, the ectoplacental cones significantly became smaller at GD9, and total area of placenta and area of spongiotrophoblast were significantly reduced at GD 13. Expression levels of Ascl2, Esx1 and Fosl1 mRNA dramatically decreased in DEHP-treated placenta at GD 13. DEHP administration disrupted labyrinth vascularization of placentas, and inhibited proliferation and induced apoptosis of placenta by the activation of caspase-3 and -8, up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein at GD 13. In conclusion, these results suggest that adverse pregnancy outcomes including low birth-weight and pregnancy loss exposed to DEHP are possibly mediated, at least in part, via the suppression of placental growth and development.

  17. Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development.

    Science.gov (United States)

    Song, You; Villeneuve, Daniel L; Toyota, Kenji; Iguchi, Taisen; Tollefsen, Knut Erik

    2017-04-18

    Molting is critical for growth, development, reproduction, and survival in arthropods. Complex neuroendocrine pathways are involved in the regulation of molting and may potentially become targets of environmental endocrine disrupting chemicals (EDCs). Based on several known ED mechanisms, a wide range of pesticides has been developed to combat unwanted organisms in food production activities such as agriculture and aquaculture. Meanwhile, these chemicals may also pose hazards to nontarget species by causing molting defects, and thus potentially affecting the health of the ecosystems. The present review summarizes the available knowledge on molting-related endocrine regulation and chemically mediated disruption in arthropods (with special focus on insects and crustaceans), to identify research gaps and develop a mechanistic model for assessing environmental hazards of these compounds. Based on the review, multiple targets of EDCs in the molting processes were identified and the link between mode of action (MoA) and adverse effects characterized to inform future studies. An adverse outcome pathway (AOP) describing ecdysone receptor agonism leading to incomplete ecdysis associated mortality was developed according to the OECD guideline and subjected to weight of evidence considerations by evolved Bradford Hill Criteria. This review proposes the first invertebrate ED AOP and may serve as a knowledge foundation for future environmental studies and AOP development.

  18. Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

    Science.gov (United States)

    Elamin, Ayssar A; Steinicke, Susanne; Oehlmann, Wulf; Braun, Yvonne; Wanas, Hanaa; Shuralev, Eduard A; Huck, Carmen; Maringer, Marko; Rohde, Manfred; Singh, Mahavir

    2017-01-01

    For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

  19. Gene therapy of cancer and development of therapeutic target gene

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Min; Kwon, Hee Chung

    1998-04-01

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

  20. The role of teacher behavior management in the development of disruptive behaviors: an intervention study with the good behavior game

    NARCIS (Netherlands)

    Leflot, G.; van Lier, P.A.C.; Onghena, P.; Colpin, H.

    2010-01-01

    The role of teacher behavior management for children's disruptive behavior development (hyperactive and oppositional behavior) was investigated using a universal classroom preventive intervention study. Five-hundred seventy children were followed from second to third grade of elementary school.

  1. Heterologous complementation of peroxisome function in yeast : The Saccharomyces cerevisiae PAS3 gene restores peroxisome biogenesis in a Hansenula polymorpha per9 disruption mutant

    NARCIS (Netherlands)

    Kiel, JAKW; KeizerGunnink, [No Value; Komori, M; Veenhuis, M; Keizer-Gunnink, I.

    1995-01-01

    PER genes are essential for the biogenesis of peroxisomes in the yeast Hansenula polymorpha. Here we describe the functional complementation of a H. polymorpha per9 disruption strain (Delta per9) by a heterologous gene. The Saccharomyces cerevisiae Pas3p, a homologue of Per9p, restored peroxisome

  2. Dynamics of the Development of Amnesia Caused by Disruption of Memory Reconsolidation by Neurotransmitter Receptors Antagonists.

    Science.gov (United States)

    Nikitin, V P; Solntseva, S V; Kozyrev, S A

    2016-03-01

    The dynamics of amnesia development under conditions of memory reconsolidation disruption by serotonin receptor antagonist methiothepin or NMDA glutamate receptor antagonist MK-801 was studied in snails trained in conventional food aversion. In 2 days after training, injection of methiothepin or MK-801 before reminder induced amnesia development. During repeated training in 3 days after amnesia induction, the skill was formed more rapidly than during the initial training. During repeated training in 10 days after administration of methiothepin and reminder, the dynamics of habit formation was similar to that during initial learning. At the same time, repeated training in 10 days after MK-801 administration and reminder did not result in long-term memory formation. Disruption of reconsolidation of conditioned food aversion memory by antagonists of serotonin or NMDA glutamate receptors led to the development of different types of amnesia that had similar strengthening gradient at the early stages, but differed by the possibility of memory formation during re-training at the late stage.

  3. Exposure to exogenous enkephalins disrupts reproductive development in the Eastern lubber grasshopper, Romalea microptera (Insecta: Orthoptera.

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar

    Full Text Available Enkephalins play a major role in reproductive physiology in crustaceans; however their role in reproductive development in insects is largely unknown. We investigated the effect of exposure to exogenous leucine-enkephalin (Leu-Enk, methionine-enkephalin (Met-Enk, and the opioid antagonist naloxone on gonad development in the Eastern lubber grasshopper, Romalea microptera. Injection of either Leu-Enk or naloxone alone significantly increased the testicular index and testicular follicular diameter in males, and the ovarian index, oocyte length, and oocyte diameter in females. In contrast, injection of Met-Enk inhibited all measures of reproductive development in both sexes. Surprisingly, co-injection of naloxone with either enkephalin enhanced the effect associated with administration of the enkephalin alone. This study clearly demonstrates the ability of enkephalins to disrupt insect sexual development and also suggests the existence of conserved enkephaline-dependent regulatory mechanisms in insects and crustaceans.

  4. Incorrect dosage of IQSEC2, a known intellectual disability and epilepsy gene, disrupts dendritic spine morphogenesis

    Science.gov (United States)

    Hinze, S J; Jackson, M R; Lie, S; Jolly, L; Field, M; Barry, S C; Harvey, R J; Shoubridge, C

    2017-01-01

    There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene (IQSEC2) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippocampal cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2, which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in

  5. LIN7A depletion disrupts cerebral cortex development, contributing to intellectual disability in 12q21-deletion syndrome.

    Directory of Open Access Journals (Sweden)

    Ayumi Matsumoto

    Full Text Available Interstitial deletion of 12q21 has been reported in four cases, which share several common clinical features, including intellectual disability (ID, low-set ears, and minor cardiac abnormalities. Comparative genomic hybridization (CGH analysis using the Agilent Human Genome CGH 180K array was performed with the genomic DNA from a two-year-old Japanese boy with these symptoms, as well as hypoplasia of the corpus callosum. Consequently, a 14 Mb deletion at 12q21.2-q21.33 (nt. 77 203 574-91 264 613 bp, which includes 72 genes, was detected. Of these, we focused on LIN7A, which encodes a scaffold protein that is important for synaptic function, as a possible responsible gene for ID, and we analyzed its role in cerebral cortex development. Western blotting analyses revealed that Lin-7A is expressed on embryonic day (E 13.5, and gradually increases in the mouse brain during the embryonic stage. Biochemical fractionation resulted in the enrichment of Lin-7A in the presynaptic fraction. Suppression of Lin-7A expression by RNAi, using in utero electroporation on E14.5, delayed neuronal migration on postnatal day (P 2, and Lin-7A-deficient neurons remained in the lower zone of the cortical plate and the intermediate zone. In addition, when Lin-7A was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed; development of these neurons was disrupted such that one half did not extend into the contralateral hemisphere after leaving the corpus callosum. Taken together, LIN7A is a candidate gene responsible for 12q21-deletion syndrome, and abnormal neuronal migration and interhemispheric axon development may contribute to ID and corpus callosum hypoplasia, respectively.

  6. LIN7A depletion disrupts cerebral cortex development, contributing to intellectual disability in 12q21-deletion syndrome.

    Science.gov (United States)

    Matsumoto, Ayumi; Mizuno, Makoto; Hamada, Nanako; Nozaki, Yasuyuki; Jimbo, Eriko F; Momoi, Mariko Y; Nagata, Koh-ichi; Yamagata, Takanori

    2014-01-01

    Interstitial deletion of 12q21 has been reported in four cases, which share several common clinical features, including intellectual disability (ID), low-set ears, and minor cardiac abnormalities. Comparative genomic hybridization (CGH) analysis using the Agilent Human Genome CGH 180K array was performed with the genomic DNA from a two-year-old Japanese boy with these symptoms, as well as hypoplasia of the corpus callosum. Consequently, a 14 Mb deletion at 12q21.2-q21.33 (nt. 77 203 574-91 264 613 bp), which includes 72 genes, was detected. Of these, we focused on LIN7A, which encodes a scaffold protein that is important for synaptic function, as a possible responsible gene for ID, and we analyzed its role in cerebral cortex development. Western blotting analyses revealed that Lin-7A is expressed on embryonic day (E) 13.5, and gradually increases in the mouse brain during the embryonic stage. Biochemical fractionation resulted in the enrichment of Lin-7A in the presynaptic fraction. Suppression of Lin-7A expression by RNAi, using in utero electroporation on E14.5, delayed neuronal migration on postnatal day (P) 2, and Lin-7A-deficient neurons remained in the lower zone of the cortical plate and the intermediate zone. In addition, when Lin-7A was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed; development of these neurons was disrupted such that one half did not extend into the contralateral hemisphere after leaving the corpus callosum. Taken together, LIN7A is a candidate gene responsible for 12q21-deletion syndrome, and abnormal neuronal migration and interhemispheric axon development may contribute to ID and corpus callosum hypoplasia, respectively.

  7. Disruption of the carA gene in Pseudomonas syringae results in reduced fitness and alters motility.

    Science.gov (United States)

    Butcher, Bronwyn G; Chakravarthy, Suma; D'Amico, Katherine; Stoos, Kari Brossard; Filiatrault, Melanie J

    2016-08-24

    Pseudomonas syringae infects diverse plant species and is widely used in the study of effector function and the molecular basis of disease. Although the relationship between bacterial metabolism, nutrient acquisition and virulence has attracted increasing attention in bacterial pathology, there is limited knowledge regarding these studies in Pseudomonas syringae. The aim of this study was to investigate the function of the carA gene and the small RNA P32, and characterize the regulation of these transcripts. Disruption of the carA gene (ΔcarA) which encodes the predicted small chain of carbamoylphosphate synthetase, resulted in arginine and pyrimidine auxotrophy in Pseudomonas syringae pv. tomato DC3000. Complementation with the wild type carA gene was able to restore growth to wild-type levels in minimal medium. Deletion of the small RNA P32, which resides immediately upstream of carA, did not result in arginine or pyrimidine auxotrophy. The expression of carA was influenced by the concentrations of both arginine and uracil in the medium. When tested for pathogenicity, ΔcarA showed reduced fitness in tomato as well as Arabidopsis when compared to the wild-type strain. In contrast, mutation of the region encoding P32 had minimal effect in planta. ΔcarA also exhibited reduced motility and increased biofilm formation, whereas disruption of P32 had no impact on motility or biofilm formation. Our data show that carA plays an important role in providing arginine and uracil for growth of the bacteria and also influences other factors that are potentially important for growth and survival during infection. Although we find that the small RNA P32 and carA are co-transcribed, P32 does not play a role in the phenotypes that carA is required for, such as motility, cell attachment, and virulence. Additionally, our data suggests that pyrimidines may be limited in the apoplastic space of the plant host tomato.

  8. Technology-enhanced program for child disruptive behavior disorders: development and pilot randomized control trial.

    Science.gov (United States)

    Jones, Deborah J; Forehand, Rex; Cuellar, Jessica; Parent, Justin; Honeycutt, Amanda; Khavjou, Olga; Gonzalez, Michelle; Anton, Margaret; Newey, Greg A

    2014-01-01

    Early onset disruptive behavior disorders are overrepresented in low-income families; yet these families are less likely to engage in behavioral parent training (BPT) than other groups. This project aimed to develop and pilot test a technology-enhanced version of one evidence-based BPT program, Helping the Noncompliant Child (HNC). The aim was to increase engagement of low-income families and, in turn, child behavior outcomes, with potential cost-savings associated with greater treatment efficiency. Low-income families of 3- to 8-year-old children with clinically significant disruptive behaviors were randomized to and completed standard HNC (n = 8) or Technology-Enhanced HNC (TE-HNC; n = 7). On average, caregivers were 37 years old; 87% were female, and 80% worked at least part-time. More than half (53%) of the youth were boys; the average age of the sample was 5.67 years. All families received the standard HNC program; however, TE-HNC also included the following smartphone enhancements: (a) skills video series, (b) brief daily surveys, (c) text message reminders, (d) video recording home practice, and (e) midweek video calls. TE-HNC yielded larger effect sizes than HNC for all engagement outcomes. Both groups yielded clinically significant improvements in disruptive behavior; however, findings suggest that the greater program engagement associated with TE-HNC boosted child treatment outcome. Further evidence for the boost afforded by the technology is revealed in family responses to postassessment interviews. Finally, cost analysis suggests that TE-HNC families also required fewer sessions than HNC families to complete the program, an efficiency that did not compromise family satisfaction. TE-HNC shows promise as an innovative approach to engaging low-income families in BPT with potential cost-savings and, therefore, merits further investigation on a larger scale.

  9. Cathepsin B gene disruption induced Leishmania donovani proteome remodeling implies cathepsin B role in secretome regulation.

    Directory of Open Access Journals (Sweden)

    Teklu Kuru Gerbaba

    Full Text Available Leishmania cysteine proteases are potential vaccine candidates and drug targets. To study the role of cathepsin B cysteine protease, we have generated and characterized cathepsin B null mutant L. donovani parasites. L. donovani cathepsin B null mutants grow normally in culture, but they show significantly attenuated virulence inside macrophages. Quantitative proteome profiling of wild type and null mutant parasites indicates cathepsin B disruption induced remodeling of L. donovani proteome. We identified 83 modulated proteins, of which 65 are decreased and 18 are increased in the null mutant parasites, and 66% (55/83 of the modulated proteins are L. donovani secreted proteins. Proteins involved in oxidation-reduction (trypanothione reductase, peroxidoxins, tryparedoxin, cytochromes and translation (ribosomal proteins are among those decreased in the null mutant parasites, and most of these proteins belong to the same complex network of proteins. Our results imply virulence role of cathepsin B via regulation of Leishmania secreted proteins.

  10. Targeted disruption of the hepatic transferrin receptor 2 gene in mice leads to iron overload.

    Science.gov (United States)

    Wallace, Daniel F; Summerville, Lesa; Subramaniam, V Nathan

    2007-01-01

    Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary hemochromatosis. Although highly expressed in liver, several studies have reported TfR2 expression in other tissues. To determine the contribution of liver expressed TfR2 in iron homeostasis, we have generated and characterized a liver-specific TfR2-knockout (KO) mouse. Liver-specific TfR2-KO mice were generated by crossing TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice with and without albumin-Cre were analyzed. Serum transferrin saturation, hepatic, and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfR1, TfR2, ferritin, and prohepcidin were analyzed by immunoblotting. Liver-specific TfR2-KO mice develop significant iron overload comparable to complete TfR2-KO mice. At all ages studied, transferrin saturation, hepatic iron concentration, and hepatic ferritin were significantly elevated. Hepatic TfR2 mRNA and protein were absent in the livers of liver-specific TfR2-KO mice, and TfR1 expression was reduced consistent with liver iron loading. At 5 weeks of age, hepcidin1 mRNA, and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice demonstrates that the liver is the primary site for TfR2 expression and activity and that liver-expressed TfR2 is required for the regulation of hepcidin1.

  11. Targeted disruption of the murine Facc gene: Towards the establishment of a mouse model for Fanconi anemia

    Energy Technology Data Exchange (ETDEWEB)

    Chen, M.; Auerbach, W.; Buchwald, M. [Hospital for Sick Childern, Toronto (Canada)] [and others

    1994-09-01

    Fanconi anemia (FA) is an autosomal recessive disease characterized by bone marrow failure, congenital malformations and predisposition to malignancies. The gene responsible for the defect in FA group C has been cloned and designated the Fanconi Anemia Complementation Group C gene (FACC). A murine cDNA for this gene (Facc) was also cloned. Here we report our progress in the establishment of a mouse model for FA. The mouse Facc cDNA was used as probe to screen a genomic library of mouse strain 129. More than twenty positive clones were isolated. Three of them were mapped and found to be overlapping clones, encompassing the genomic region from exon 8 to the end of the 3{prime} UTR of the mouse cDNA. A targeting vector was constructed using the most 5{prime} mouse genomic sequence available. The end result of the homologous recombination is that exon 8 is deleted and the neo gene is inserted. The last exon, exon 14, is essential for the complementing function of the FACC gene product; the disruption in the middle of the murine Facc gene should render this locus biologically inactive. This targeting vector was linearized and electroporated into R1 embryonic stem (ES) cells which were derived from the 129 mouse. Of 102 clones screened, 19 positive cell lines were identified. Four targeted cell lines have been used to produce chimeric mice. 129-derived ES cells were aggregated ex vivo into the morulas derived from CD1 mice and then implanted into foster mothers. 22 chimeras have been obtained. Moderately and strongly chimeric mice have been bred to test for germline transmission. Progeny with the expected coat color derived from 2 chimeras are currently being examined to confirm transmission of the targeted allele.

  12. EAAC1 Gene Deletion Increases Neuronal Death and Blood Brain Barrier Disruption after Transient Cerebral Ischemia in Female Mice

    Directory of Open Access Journals (Sweden)

    Bo Young Choi

    2014-10-01

    Full Text Available EAAC1 is important in modulating brain ischemic tolerance. Mice lacking EAAC1 exhibit increased susceptibility to neuronal oxidative stress in mice after transient cerebral ischemia. EAAC1 was first described as a glutamate transporter but later recognized to also function as a cysteine transporter in neurons. EAAC1-mediated transport of cysteine into neurons contributes to neuronal antioxidant function by providing cysteine substrates for glutathione synthesis. Here we evaluated the effects of EAAC1 gene deletion on hippocampal blood vessel disorganization after transient cerebral ischemia. EAAC1−/− female mice subjected to transient cerebral ischemia by common carotid artery occlusion for 30 min exhibited twice as much hippocampal neuronal death compared to wild-type female mice as well as increased reduction of neuronal glutathione, blood–brain barrier (BBB disruption and vessel disorganization. Pre-treatment of N-acetyl cysteine, a membrane-permeant cysteine prodrug, increased basal glutathione levels in the EAAC1−/− female mice and reduced ischemic neuronal death, BBB disruption and vessel disorganization. These findings suggest that cysteine uptake by EAAC1 is important for neuronal antioxidant function under ischemic conditions.

  13. Formation of Highly Twisted Ribbons in a Carboxymethylcellulase Gene-Disrupted Strain of a Cellulose-Producing Bacterium

    Science.gov (United States)

    Sugano, Yasushi; Shoda, Makoto; Sakakibara, Hitoshi; Oiwa, Kazuhiro; Tuzi, Satoru; Imai, Tomoya; Sugiyama, Junji; Takeuchi, Miyuki; Yamauchi, Daisuke

    2013-01-01

    Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state 13C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains. PMID:23243308

  14. Erosion of newly developed CFCs and Be under disruption heat loads

    Science.gov (United States)

    Nakamura, K.; Akiba, M.; Araki, M.; Dairaku, M.; Sato, K.; Suzuki, S.; Yokoyama, K.; Linke, J.; Duwe, R.; Bolt, H.; Roedig, M.

    1996-10-01

    An evaluation of the erosion under disruption heat loads is very important to the lifetime prediction of divertor armour tiles of next fusion devices such as ITER. In particular, erosion data on CFCs (carbon fiber reinforced composites) and beryllium (Be) as the armour materials is urgently required in the ITER design. For CFCs, high heat flux experiments on the newly developed CFCs with high thermal conductivity have been performed under the heat flux of around 800-2000 MW/m 2 and the pulse length of 2-5 ms in JAERI electron beam irradiation systems (JEBIS). As a result, the weight losses of B 4C doped CFCs after heating were almost same to those of the non doped CFC up to 5 wt% boron content. For Be, we have carried out our first disruption experiments on S65/C grade Be specimens in the Juelich divertor test facility in hot cells (JUDITH) facility as a frame work of the J—EU collaboration. The heating conditions were heat loads of 1250-5000 MW/m 2 for 2-8 ms, and the heated area was 3 × 3 mm 2. As a result, the protuberances of the heated area of Be were observed under the lower heat flux.

  15. Transplacental exposure to inorganic arsenic at a hepatocarcinogenic dose induces fetal gene expression changes in mice indicative of aberrant estrogen signaling and disrupted steroid metabolism

    Science.gov (United States)

    Liu, Jie; Xie, Yaxiong; Cooper, Ryan; Ducharme, Danica M.K.; Tennant, Raymond; Diwan, Bhalchandra A.; Waalkes, Michael P.

    2009-01-01

    Exposure to inorganic arsenic in utero in C3H mice produces hepatocellular carcinoma in male offspring when they reach adulthood. To help define the molecular events associated with the fetal onset of arsenic hepatocarcinogenesis, pregnant C3H mice were given drinking water containing 0 (control) or 85 ppm arsenic from day 8 to 18 of gestation. At the end of the arsenic exposure period, male fetal livers were removed and RNA isolated for microarray analysis using 22K oligo chips. Arsenic exposure in utero produced significant (p inorganic arsenic during a critical period in development significantly alters the expression of various genes encoding estrogen signaling and steroid or methionine metabolism. These alterations could disrupt genetic programming at the very early life-stage, which could impact tumor formation much later in adulthood. PMID:17350061

  16. Short-term exposure of arsenite disrupted thyroid endocrine system and altered gene transcription in the HPT axis in zebrafish.

    Science.gov (United States)

    Sun, Hong-Jie; Li, Hong-Bo; Xiang, Ping; Zhang, Xiaowei; Ma, Lena Q

    2015-10-01

    Arsenic (As) pollution in aquatic environment may adversely impact fish health by disrupting their thyroid hormone homeostasis. In this study, we explored the effect of short-term exposure of arsenite (AsIII) on thyroid endocrine system in zebrafish. We measured As concentrations, As speciation, and thyroid hormone thyroxine levels in whole zebrafish, oxidative stress (H2O2) and damage (MDA) in the liver, and gene transcription in hypothalamic-pituitary-thyroid (HPT) axis in the brain and liver tissues of zebrafish after exposing to different AsIII concentrations for 48 h. Result indicated that exposure to AsIII increased inorganic As in zebrafish to 0.46-0.72 mg kg(-1), induced oxidative stress with H2O2 being increased by 1.4-2.5 times and caused oxidative damage with MDA being augmented by 1.6 times. AsIII exposure increased thyroxine levels by 1.3-1.4 times and modulated gene transcription in HPT axis. Our study showed AsIII caused oxidative damage, affected thyroid endocrine system and altered gene transcription in HPT axis in zebrafish. Published by Elsevier Ltd.

  17. Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

    Science.gov (United States)

    Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

    2017-08-01

    CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

  18. Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanisms and unique phenotypic consequences.

    Science.gov (United States)

    Bhatia, Shipra; Kleinjan, Dirk A

    2014-07-01

    The precise control of gene expression programs is crucial for the establishment of the diverse gene activity patterns required for the correct development, patterning and differentiation of the myriad of cell types within an organism. The crucial importance of non-coding regions of the genome in the control of gene regulation is well established and depends on a diverse group of sequence fragments called cis-regulatory elements that reside in these regions. Advances in novel genome-wide techniques have greatly increased the ability to identify potential regulatory elements. In contrast, their functional characterisation and the determination of their diverse modes of action remain a major bottleneck. Greater knowledge of gene expression control is of major importance for human health as disruption of gene regulation has become recognised as a significant cause of human disease. Appreciation of the role of cis-regulatory polymorphism in natural variation and susceptibility to common disease is also growing. While novel techniques such as GWAS and NGS provide the ability to collect large genomic datasets, the challenge for the twenty-first century will be to extract the relevant sequences and how to investigate the functional consequences of disease-associated changes. Here, we review how studies of transcriptional control at selected paradigm disease gene loci have revealed general principles of cis-regulatory logic and regulatory genome organisation, yet also demonstrate how the variety of mechanisms can combine to result in unique phenotypic outcomes. Integration of these principles with the emerging wealth of genome-wide data will provide enhanced insight into the workings of our regulatory genome.

  19. Sex-specific differences in natriuretic peptide and nitric oxide synthase expression in ANP gene-disrupted mice.

    Science.gov (United States)

    Wong, Philip G; Armstrong, David W J; Tse, M Yat; Brander, Emily P A; Pang, Stephen C

    2013-02-01

    Sex-specific differences in hormone-mediated gene regulation may influence susceptibility to cardiac hypertrophy, a primary risk factor for cardiovascular disease. Under hormonal influence, natriuretic peptide (NP) and nitric oxide synthase (NOS) systems modulate cardio-protective gene programs through common downstream production of cyclic guanosine 3'-5' monophosphate (cGMP). Ablation of either system can adversely affect cardiac adaptation to stresses and insults. This study elucidates sex-specific differences in cardiac NP and NOS system gene expression and assesses the impact of the estrous cycle on these systems using the atrial natriuretic peptide gene-disrupted (ANP(-/-)) mouse model. Left ventricular expression of the NP and NOS systems was analyzed using real-time quantitative polymerase chain reaction in 13- to 16-week-old male, proestrous and estrous female ANP(+/+) and ANP(-/-) mice. Left ventricular and plasma cGMP levels were measured to assess the convergent downstream effects of the NP and NOS systems. Regardless of genotype, males had higher expression of the NP system while females had higher expression of the NOS system. In females, transition from proestrus to estrus lowered NOS system expression in ANP(+/+) mice while the opposite was observed in ANP(-/-) mice. No significant changes in left ventricular cGMP levels across gender and genotype were observed. Significantly lower plasma cGMP levels were observed in ANP(-/-) mice compared to ANP(+/+) mice. Regardless of genotype, sex-specific differences in cardiac NP and NOS system expression exist, each sex enlisting a predominant system to conserve downstream cGMP. Estrous cycle-mediated alterations in NOS system expression suggests additional hormone-mediated gene regulation in females.

  20. Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Yu L

    2014-12-01

    Full Text Available Lan Yu, Xiaoli Wang, Da Zhu, Wencheng Ding, Liming Wang, Changlin Zhang, Xiaohui Jiang, Hui Shen, Shujie Liao, Ding Ma, Zheng Hu, Hui Wang Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China Abstract: High-risk human papillomavirus (HPV, especially HPV16, is considered a main causative agent of cervical cancer. Upon HPV infection, the viral oncoprotein E6 disrupts the host tumor-suppressor protein p53, thus promoting malignant transformation of normal cervical cells. Here, we used the newly developed programmable ribonucleic acid-guided clustered regularly interspaced short palindromic repeat (CRISPR/Cas system to disrupt the HPV16 E6 gene. We showed that HPV16 E6 deoxyribonucleic acid was cleaved at specific sites, leading to apoptosis and growth inhibition of HPV16-positive SiHa and CaSki cells, but not HPV-negative C33A or human embryonic kidney 293 cells. We also observed downregulation of the E6 protein and restoration of the p53 protein. These data proved that the HPV16 E6 ribonucleic acid-guided CRISPR/Cas system might be an effective therapeutic agent in treating HPV infection-related cervical malignancy. Keywords: CRISPR/Cas system, E6, p53, SiHa, CaSki, cervical cancer

  1. Disruption of Imprinted Genes at Chromosome Region 11p15.5 in Paediatric Rhabdomyosarcoma

    Directory of Open Access Journals (Sweden)

    John Anderson

    1999-10-01

    Full Text Available Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH at chromosome region 11pl5.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2, H19, p57KIP2. We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3—FKHR or PAX7—FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57KIP2 was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57KIP2 expression, there was no evidence for inactivating mutations, suggesting that p57KIP2 is not a tumour suppressor in rhabdomyosarcoma.

  2. Small Molecule Disrupts Abnormal Gene Fusion Associated with Leukemia | Center for Cancer Research

    Science.gov (United States)

    Rare chromosomal abnormalities, called chromosomal translocations, in which part of a chromosome breaks off and becomes attached to another chromosome, can result in the generation of chimeric proteins. These aberrant proteins have unpredictable, and sometimes harmful, functions, including uncontrolled cell growth that can lead to cancer. One type of translocation, in which a portion of the gene encoding nucleoporin 98 (NUP98)—one of about 50 proteins comprising the nuclear pore complex through which proteins are shuttled into and out of the nucleus—fuses with another gene, has been shown to result in improper histone modifications. These abnormalities alter the gene expression patterns of certain types of hematopoietic, or blood-forming, stem cells, resulting primarily in overexpression of the Hoxa7, Hoxa9,and Hoxa10 genes. NUP98 chromosomal translocations have been associated with many types of leukemia, including acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia in blast crisis (CML-bc), and myelodysplastic syndrome (MDS).

  3. Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice

    DEFF Research Database (Denmark)

    Xu, T; Bianco, P; Fisher, L W

    1998-01-01

    The resilience and strength of bone is due to the orderly mineralization of a specialized extracellular matrix (ECM) composed of type I collagen (90%) and a host of non-collagenous proteins that are, in general, also found in other tissues. Biglycan (encoded by the gene Bgn) is an ECM proteoglycan...

  4. Disruption of Arabidopsis reticulon gene rtnlb16 results in chloroplast dysfunction and oxidative stress

    OpenAIRE

    Tarasenko V.I.; E.Yu. Garnik; A.I. Katyshev; I.Yu. Subota; Yu.M. Konstantinov

    2012-01-01

    Reticulons (RTNs) are endoplasmic reticulum (ER)-localized proteins that have recently attracted much attention. RTNs are ubiquitous proteins present in all eukaryotic organisms examined so far. In animal and yeast, in which knowledge of this protein family is more advanced, RTNs are involved in numerous cellular processes such as apoptosis, cell division and intracellular trafficking. Up to now, a little attention has been paid to their plant counterparts, RTNLBs. Meanwhile, gene search acro...

  5. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  6. Disruption of the 37-kDa/67-kDa laminin receptor gene in bovine ...

    African Journals Online (AJOL)

    The 37-kDa/67-kDa laminin receptor (LRP/LR), also known as ribosomal protein SA (RPSA), acts as a cell surface receptor for prions and plays an important role in internalization of cellular prion protein. In this study, we knocked out the part of prion binding sites (aa 161-205) by gene targeting in the bovine fetal fibroblasts ...

  7. Disruption of the gaa Gene in Zebrafish Fails to Generate the Phenotype of Classical Pompe Disease.

    Science.gov (United States)

    Wu, Jing; Yang, Yi; Sun, Chengjun; Sun, Shaoyang; Li, Qiang; Yao, Yuxiao; Fei, Fei; Lu, Lingeng; Chang, Zhuo; Zhang, Wenting; Wang, Xu; Luo, Feihong

    2017-01-01

    The underlying pathogenic lesions of glycogen storage disease type II (GSD II) and the diversity of this disease among different species are still under exploration. Thus, we created an acid alpha-glucosidase (gaa) gene-mutated zebrafish model of GSD II and examined the sequential pathogenic changes. gaa mRNA and protein expression, enzymatic activity, and lysosomal glycogen accumulation were assessed, and the phenotypic changes were compared between wild-type (WT) and gaa-mutated zebrafish. The presence of a Δ13 frameshift mutation in the gaa gene was confirmed at both the DNA and transcribed mRNA levels by Sanger sequencing. The relative amount of gaa mRNA was decreased before 2 days postfertilization (dpf), after which it unexpectedly increased in the mutant compared with the WT zebrafish. Consistent with the mRNA expression, the Gaa enzymatic activity in the mutant was downregulated before 3 dpf, while the Gaa protein level was slightly decreased at 4 dpf and was maintained at a consistent level in the adult gaa mutant muscle tissue. However, more than half of the adult mutant zebrafish exhibited excessive glycogen accumulation in the liver and muscles, along with the presence of autophagosomes, as determined by transmission electron microscopy. Thus, we have successfully generated a frameshift mutation in the gaa gene in zebrafish. The unique gaa gene expression changes and mild GSD II features during the adult stage strongly indicate the existence of species-specific differences, as well as an underlying compensatory network, which may warrant further examination.

  8. Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β

    OpenAIRE

    Punia, Sohan; Rumery, Kyle K.; Yu, Elizabeth A.; Christopher M Lambert; Notkins, Abner L.; David R Weaver

    2012-01-01

    Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molec...

  9. Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

    Science.gov (United States)

    Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

    2017-09-07

    The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  10. DISRUPTION OF ARABIDOPSIS RETICULON GENE RTNLB16 RESULTS IN CHLOROPLAST DYSFUNCTION AND OXIDATIVE STRESS

    Directory of Open Access Journals (Sweden)

    Tarasenko V.I.

    2012-08-01

    Full Text Available Reticulons (RTNs are endoplasmic reticulum (ER-localized proteins that have recently attracted much attention. RTNs are ubiquitous proteins present in all eukaryotic organisms examined so far. In animal and yeast, in which knowledge of this protein family is more advanced, RTNs are involved in numerous cellular processes such as apoptosis, cell division and intracellular trafficking. Up to now, a little attention has been paid to their plant counterparts, RTNLBs. Meanwhile, gene search across sequenced genomes revealed that the RTN gene family is more diverse and numerous in plants than in animals and yeasts, which possibly suggests existence of functions specific for plant RTNs. Recently, the localization in different ER regions was shown for two members of plant reticulon family. The location in close proximity to chloroplast membrane was revealed for one of RTNLBs, which is argument in favor of its role in interorganellar interactions. In spite of growing interest towards to plant RTNs, there are no investigations devoted to insertion mutagenesis of genes encoding these proteins. We have genotyped an Arabidopsis line containing T-DNA insertion in RTNLB16 gene encoding uncharacterized member of RTNLB family. The obtained homozygous plants have marked phenotype expressed in a decreased growth rate and a pale-green leaf color. The leaf total chlorophyll content as well as the chlorophyll a/b ratio was significantly lower in mutant plants. It is interesting to note that the extent of phenotypic expression depended on a light intensity. The growth rate of wild-type and mutant plants was the same in low light conditions. The growth rate was significantly decreased and chlorophyll content was 3-5-fold lower in mutant plants growing under moderate light conditions. The growing of plants under high light conditions led to halted growth and death of mutants on the seedling stage. The demonstrated phenotype probably points out to a chloroplast

  11. DPP6 gene disruption in a family with Gilles de la Tourette syndrome.

    Science.gov (United States)

    Prontera, Paolo; Napolioni, Valerio; Ottaviani, Valentina; Rogaia, Daniela; Fusco, Carmela; Augello, Bartolomeo; Serino, Domenico; Parisi, Valentina; Bernardini, Laura; Merla, Giuseppe; Cavanna, Andrea E; Donti, Emilio

    2014-10-01

    Gilles de la Tourette syndrome (TS) is a neurodevelopmental disorder characterized by multiple motor and vocal tics, frequently associated with psychiatric co-morbidities. Despite the significant level of heritability, the genetic architecture of TS still remains elusive. Herein, we investigated an Italian family where an 8-year-old boy, his father, and paternal uncle have a diagnosis of TS. Array-CGH and high resolution SNP-array analyses revealed a heterozygous microdeletion of ∼135 kb at the 7q36.2 locus in the proband and his father. Fluorescent in situ hybridization and quantitative PCR (qPCR) analyses confirmed the presence of the alteration also in the paternal uncle. The deletion selectively involves the first exon of the DPP6 gene, leading to a down-regulation of its expression, as demonstrated by the reduced messenger RNA (mRNA) levels assessed by RT-qPCR. The DPP6 gene encodes for a type II membrane glycoprotein expressed predominantly in the central nervous system. To date, a de novo DPP6 exonic duplication, of uncertain significance, was reported in one patient with TS. Moreover, the DPP6 gene has been implicated in the pathogenesis of autism spectrum disorder (ASD) and, notably, in haloperidol-induced dyskinesia. This first familial case provides evidence for association between DPP6 haploinsufficiency and TS, further suggesting a plausible molecular link between TS and ASD, and might shed some light on the efficacy and tolerability profiles of antidopaminergic agents used for tic management, thus prompting further studies on a larger cohort of patients.

  12. The Growth Hormone Receptor Gene-Disrupted (GHR-KO) Mouse Fails to Respond to an Intermittent Fasting (IF) Diet

    Science.gov (United States)

    Arum, Oge; Bonkowski, Michael S.; Rocha, Juliana S.; Bartke, Andrzej

    2009-01-01

    SUMMARY The interaction of longevity-conferring genes with longevity-conferring diets is poorly understood. The growth hormone receptor gene-disrupted (GHR-KO) mouse is long-lived; and this longevity is not responsive to 30% caloric restriction (CR), in contrast to wild-type animals from the same strain. To determine whether this may have been limited to a particular level of dietary restriction (DR), we subjected GHR-KO mice to a different dietary restriction regimen, an intermittent fasting (IF) diet. The IF diet increased the survivorship and improved insulin sensitivity of normal males, but failed to affect either parameter in GHR-KO mice. From the results of two paradigms of dietary restriction we postulate that GHR-KO mice would be resistant to any manner of DR; potentially due to their inability to further enhance insulin sensitivity. Insulin sensitivity may be a mechanism and/or a marker of the lifespan-extending potential of an intervention. PMID:19747233

  13. The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors.

    Science.gov (United States)

    Dougherty, Joseph D; Maloney, Susan E; Wozniak, David F; Rieger, Michael A; Sonnenblick, Lisa; Coppola, Giovanni; Mahieu, Nathaniel G; Zhang, Juliet; Cai, Jinlu; Patti, Gary J; Abrahams, Brett S; Geschwind, Daniel H; Heintz, Nathaniel

    2013-02-13

    The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders.

  14. Putative effects of endocrine disrupters on pubertal development in the human

    DEFF Research Database (Denmark)

    Teilmann, Grete; Juul, Anders; Skakkebaek, Niels E

    2002-01-01

    -called endocrine disrupters. Precocious puberty has been described in several case reports of accidental exposure to oestrogenic compounds in cosmetic products, food and pharmaceuticals. Local epidemics of premature thelarche have also been suggested to be linked to endocrine disrupters. Children adopted from...

  15. Construction of a dextran-free Leuconostoc citreum mutant by targeted disruption of the dextransucrase gene.

    Science.gov (United States)

    Jin, Q; Li, L; Kim, Y J; Han, N S

    2014-10-01

    Leuconostoc citreum is an important lactic acid bacterium in fermented foods, but dextran production often causes undesired ropiness. To prevent this side effect, a dextran-free mutant needs to be created. Homologous recombination of the dextransucrase gene (dsrC) was conducted using a segregationally unstable plasmid, pCBM32-DSUDs. A mutant was obtained on sucrose agar medium, and a site-specific insertional inactivation in the gene was confirmed. When cultured in sucrose medium, the mutant strain produced no dextransucrase or dextran. Additionally, it showed a longer lag phase (9 h) than the wild-type strain (3 h), providing new insights into the role of dextransucrase in carbohydrate metabolism of Leuconostoc. In this study, a dextransucrase knockout mutant was constructed. It was found that Leuc. citreum dextransucrase not only synthesizes dextran for cell protection but also provides fructose as an important carbon source for cell growth. This knockout mutation was generated for the first time in Leuc. citreum. The dextran-free mutant has the potential to be used for various industrial purposes, including as a starter culture for production of nonviscous fermented foods and as a dextran-free host for production of recombinant proteins. © 2014 The Society for Applied Microbiology.

  16. Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

    Science.gov (United States)

    Kozol, Robert A.; Cukier, Holly N.; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J.; Whitehead, Patrice L.; Haines, Jonathan L.; Gilbert, John R.; Cuccaro, Michael L.; Martin, Eden R.; Baker, James D.; Buxbaum, Joseph D.; Pericak-Vance, Margaret A.; Dallman, Julia E.

    2015-01-01

    Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1–2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

  17. The Science of Neglect: The Persistent Absence of Responsive Care Disrupts the Developing Brain. Working Paper 12

    Science.gov (United States)

    National Scientific Council on the Developing Child, 2012

    2012-01-01

    Young children who experience severe deprivation or neglect can experience a range of negative consequences. Neglect can delay brain development, impair executive function skills, and disrupt the body's stress response. This working paper from the National Scientific Council on the Developing Child explains why neglect is so harmful in the…

  18. Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene

    Directory of Open Access Journals (Sweden)

    DeGregori James V

    2003-01-01

    Full Text Available Abstract Background Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission. Results Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression. Conclusions Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

  19. Equatorial Spread F Development/Disruption under Disturbance Electric Fields during Some Recent Intense Magnetic Storms.

    Science.gov (United States)

    Abdu, M. A.; Paula, E. R.; Batista, I. S.; Reinisch, B. W.; Denardini, C. M.; Sobral, J. H.

    2006-05-01

    Equatorial spread F (ESF) and associated plasma bubble irregularity development can be initiated or inhibited under disturbance electric fields associated with magnetic storms. Case studies of ESF intensification/inhibition under intense storm conditions are rare, however. We have addressed this question using the data collected from the Brazilian network of Digisondes, a VHF radar and GPS scintillation receivers, complemented by Digisonde data from Jicamarca and satellite born measurements. Dawn-dusk electric fields penetrating to equatorial latitude in the early phase of a storm event cause disruption of ESF development under a nightside westward electric field, that causes a rapid descent of the F layer. Large intensity prompt penetration electric fields occurring on the evening/post sunset ionosphere cause rapid uplifts of the F layer, and existing ESF patch structures seen by the radar to move to higher altitudes often exceeding the limits of the instruments (>1300 km) so that possible generation of irregularities by disturbance electric fields can only be detected by scintillation receivers. Disturbance dynamo electric fields are found to strongly influence the ESF development, which is inhibited during post sunset hours while subject to intensification during post midnight hours. The possible role of a disturbance trans-equatorial wind in suppressing the ESF generation is also examined

  20. DEVELOPMENT OF DISRUPTIVE BEHAVIORS IN YOUNG CHILDREN: A PROSPECTIVE POPULATION-BASED COHORT STUDY

    Science.gov (United States)

    BAILLARGEON, RAYMOND H.; MORISSET, ALEXANDRE; KEENAN, KATE; NORMAND, CLAUDE L.; SÉGUIN, JEAN R.; JAPEL, CHRISTA; CAO, GUANQIONG

    2013-01-01

    We know relatively little about the development of disruptive behaviors (DBs), and gender differences therein. The objective of this study was to describe the continuity and discontinuity in the degree to which young children in the general population are reported to exhibit specific DBs over time. Data came from the Québec Longitudinal Study of Child Development. First, the results show that relatively few children exhibit DBs on a frequent basis at 41 months of age. Second, the results show that a majority of children who exhibit a particular DB on a frequent basis at 41 months of age did not do so 1 year earlier. In addition, a majority of children who exhibited a particular DB on a frequent basis at 29 months of age no longer do so 1 year later. Third, gender differences in DBs (boys > girls) are either emerging or at least increasing in magnitude between 29 and 41 months of age. Consistent with the canalization of the behavioral development principle, children who exhibited DBs on a frequent basis at 29 months of age are less likely to stop doing so in the following year if they had exhibited the same behaviors at 17 months of age. PMID:28079905

  1. Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis).

    Science.gov (United States)

    Ortega, Encarnación; Martínez-García, Pedro J; Dicenta, Federico; Egea, José

    2010-06-01

    A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.

  2. Disruptions in valine degradation affect seed development and germination in Arabidopsis.

    Science.gov (United States)

    Gipson, Andrew B; Morton, Kyla J; Rhee, Rachel J; Simo, Szabolcs; Clayton, Jack A; Perrett, Morgan E; Binkley, Christiana G; Jensen, Erika L; Oakes, Dana L; Rouhier, Matthew F; Rouhier, Kerry A

    2017-06-01

    We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched-chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3-hydroxyisobutyryl-CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched-chain amino acid catabolism plays in seed development and amino acid homeostasis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  3. Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

    Science.gov (United States)

    Delneri, D; Gardner, D C; Bruschi, C V; Oliver, S G

    1999-11-01

    By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect. Copyright 1999 John Wiley & Sons, Ltd.

  4. The deletion of Math5 disrupts retinal blood vessel and glial development in mice

    Science.gov (United States)

    Edwards, Malia M.; McLeod, D. Scott; Li, Renzhong; Grebe, Rhonda; Bhutto, Imran; Mu, Xiuqian; Lutty, Gerard A.

    2011-01-01

    Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5−/− mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5−/− mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5−/− mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5−/− mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5−/− retina. In addition, GFAP+ Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5−/− mice is associated with a lack of retinal vascular development. PMID:22200487

  5. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult.

    Directory of Open Access Journals (Sweden)

    Vinicius S Carreira

    Full Text Available The Developmental Origins of Health and Disease (DOHaD Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR, either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.

  6. RNA interference can be used to disrupt gene function in tardigrades

    OpenAIRE

    Tenlen, Jennifer R.; McCaskill, Shaina; Goldstein, Bob

    2012-01-01

    How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are als...

  7. A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly.

    Science.gov (United States)

    Pichon, Bruno; Vankerckhove, Sophie; Bourrouillou, Georges; Duprez, Laurence; Abramowicz, Marc J

    2004-05-01

    Primary microcephaly (microcephalia vera) is a developmental abnormality resulting in a small brain, with mental retardation. It is usually transmitted as an autosomal recessive trait, and six loci have been reported to date. We analyzed a translocation breakpoint previously reported in a patient with apparently sporadic primary microcephaly, at 1q31, where locus MCPH5 maps. The patient was lost to follow-up, and we sampled a maternal aunt who carried the familial translocation. FISH analyses showed that the insert of BAC clone RP11-32D17 spanned the breakpoint. The breakpoint was further located within a fragment of this insert corresponding to intron 17 of the ASPM gene, resulting in a predicted transcript truncated of more than half of its coding sequence. It is very likely that the proband carried a second ASPM mutation in trans, but he was not available for sampling and hence we could not confirm this hypothesis. Our observation adds to the mutation spectrum of ASPM in primary microcephaly, and is to our knowledge the second example of a constitutional, reciprocal translocation responsible for a bona fide autosomal recessive phenotype.

  8. Development of integrated core disruptive accident analysis code for FBR - ASTERIA-FBR

    Energy Technology Data Exchange (ETDEWEB)

    Ishizu, T.; Endo, H.; Tatewaki, I.; Yamamoto, T. [Japan Nuclear Energy Safety Organization JNES, Toranomon Towers Office, 4-1-28, Toranomon, Minato-ku, Tokyo (Japan); Shirakawa, N. [Inst. of Applied Energy IAE, Shimbashi SY Bldg., 14-2 Nishi-Shimbashi 1-Chome, Minato-ku, Tokyo (Japan)

    2012-07-01

    The evaluation of consequence at the severe accident is the most important as a safety licensing issue for the reactor core of liquid metal cooled fast breeder reactor (LMFBR), since the LMFBR core is not in an optimum condition from the viewpoint of reactivity. This characteristics might induce a super-prompt criticality due to the core geometry change during the core disruptive accident (CDA). The previous CDA analysis codes have been modeled in plural phases dependent on the mechanism driving a super-prompt criticality. Then, the following event is calculated by connecting different codes. This scheme, however, should introduce uncertainty and/or arbitrary to calculation results. To resolve the issues and obtain the consistent calculation results without arbitrary, JNES is developing the ASTERIA-FBR code for the purpose of providing the cross-check analysis code, which is another required scheme to confirm the validity of the evaluation results prepared by applicants, in the safety licensing procedure of the planned high performance core of Monju. ASTERIA-FBR consists of the three major calculation modules, CONCORD, dynamic-GMVP, and FEMAXI-FBR. CONCORD is a three-dimensional thermal-hydraulics calculation module with multi-phase, multi-component, and multi-velocity field model. Dynamic-GMVP is a space-time neutronics calculation module. FEMAXI-FBR calculates the fuel pellet deformation behavior and fuel pin failure behavior. This paper describes the needs of ASTERIA-FBR development, major module outlines, and the model validation status. (authors)

  9. Gene Regulation Networks for Modeling Drosophila Development

    Science.gov (United States)

    Mjolsness, E.

    1999-01-01

    This chapter will very briefly introduce and review some computational experiments in using trainable gene regulation network models to simulate and understand selected episodes in the development of the fruit fly, Drosophila Melanogaster.

  10. Gene expression profiling during murine tooth development

    Directory of Open Access Journals (Sweden)

    Maria A dos Santos silva Landin

    2012-07-01

    Full Text Available The aim of this study was to describe the expression of genes, including ameloblastin (Ambn, amelogenin X chromosome (Amelx and enamelin (Enam during early (pre-secretory tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24h intervals, starting at the eleventh embryonic day (E11.5 and up to the seventh day after birth (P7. The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx and Enam. Microarray results where validated using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR, and translated proteins identified by Western blotting. In situ localisation of the Ambn, Amelx and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially (p ≤0.05 expressed (DE genes.Microarray results showed a total of 4362 genes including Ambn, Amelx and Enam to be significant differentially expressed throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0 increasing after birth (P1-P7. Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. The mRNAs expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around thirty-five genes were associated with fifteen transcription factors.

  11. Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis.

    Science.gov (United States)

    Zheng, Huanquan; Rowland, Owen; Kunst, Ljerka

    2005-05-01

    In the absence of cell migration, plant architecture is largely determined by the direction and extent of cell expansion during development. In this report, we show that very-long-chain fatty acid (VLCFA) synthesis plays an essential role in cell expansion. The Arabidopsis thaliana eceriferum10 (cer10) mutants exhibit severe morphological abnormalities and reduced size of aerial organs. These mutants are disrupted in the At3g55360 gene, previously identified as a gene coding for enoyl-CoA reductase (ECR), an enzyme required for VLCFA synthesis. The absence of ECR activity results in a reduction of cuticular wax load and affects VLCFA composition of seed triacylglycerols and sphingolipids, demonstrating in planta that ECR is involved in all VLCFA elongation reactions in Arabidopsis. Epidermal and seed-specific silencing of ECR activity resulted in a reduction of cuticular wax load and the VLCFA content of seed triacylglycerols, respectively, with no effects on plant morphogenesis, suggesting that the developmental phenotypes arise from abnormal sphingolipid composition. Cellular analysis revealed aberrant endocytic membrane traffic and defective cell expansion underlying the morphological defects of cer10 mutants.

  12. Factor for adipocyte differentiation 158 gene disruption prevents the body weight gain and insulin resistance induced by a high-fat diet.

    Science.gov (United States)

    Hayashi, Takahiro; Nozaki, Yuriko; Nishizuka, Makoto; Ikawa, Masahito; Osada, Shigehiro; Imagawa, Masayoshi

    2011-01-01

    To clarify the molecular mechanism of adipocyte differentiation, we previously isolated a novel gene, factor for adipocyte differentiation (fad) 158, whose expression was induced during the earliest stages of adipogenesis, and its product was localized to the endoplasmic reticulum. We found that the knockdown of fad158 expression prevented the differentiation of 3T3-L1 cells into adipocytes. In addition, over-expression of fad158 promoted the differentiation of NIH-3T3 cells, which do not usually differentiate into adipocytes. Although these findings strongly suggest that fad158 has a crucial role in regulating adipocyte differentiation, the physiological role of the gene is still unclear. In this study, we generated mice in which fad158 expression was deleted. The fad158-deficient mice did not show remarkable changes in body weight or the weight of white adipose tissue on a chow diet, but had significantly lower body weights and fat mass than wild-type mice when fed a high-fat diet. Furthermore, although the disruption of fad158 did not influence insulin sensitivity on the chow diet, it improved insulin resistance induced by the high-fat diet. These results indicate that fad158 is a key factor in the development of obesity and insulin resistance caused by a high-fat diet.

  13. Disruption of the 37-kDa/67-kDa laminin receptor gene in bovine ...

    African Journals Online (AJOL)

    enoh

    2012-03-22

    Mar 22, 2012 ... State Key Laboratories for Agrobiotechnology, Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture,. National Animal ... disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in .... With the development of cloning techniques, transgenic animals can be ...

  14. Development of a Biosensor for Identifying Novel Endocrine-Disrupting Chemicals

    National Research Council Canada - National Science Library

    Standley, Laurel J

    2008-01-01

    Substantial evidence indicates that endocrine disrupting chemicals (EDCs) particularly those that interact with the estrogen receptor may play a role in reproduction and hormonal cancers in humans and animals...

  15. Disruptive Behavior Scale Professed by Students (DBS-PS): Development and Validation

    OpenAIRE

    Veiga, Feliciano

    2008-01-01

    This study presents both the construction procedures and the results obtained with a 16- items Disruptive Behavior Scale Professed by Students (DBS-PS) for Portuguese students. The sample was made of 915 subjects from the 7th, the 8th, and the 9th grades. Results obtained with that self-report instrument were analyzed, and measures of reliability and of construct and concurrent discrimination were estimated. Three specific factors were identified for the school disruption throu...

  16. ESRP1 Mutations Cause Hearing Loss due to Defects in Alternative Splicing that Disrupt Cochlear Development.

    Science.gov (United States)

    Rohacek, Alex M; Bebee, Thomas W; Tilton, Richard K; Radens, Caleb M; McDermott-Roe, Chris; Peart, Natoya; Kaur, Maninder; Zaykaner, Michael; Cieply, Benjamin; Musunuru, Kiran; Barash, Yoseph; Germiller, John A; Krantz, Ian D; Carstens, Russ P; Epstein, Douglas J

    2017-11-06

    Alternative splicing contributes to gene expression dynamics in many tissues, yet its role in auditory development remains unclear. We performed whole-exome sequencing in individuals with sensorineural hearing loss (SNHL) and identified pathogenic mutations in Epithelial Splicing-Regulatory Protein 1 (ESRP1). Patient-derived induced pluripotent stem cells showed alternative splicing defects that were restored upon repair of an ESRP1 mutant allele. To determine how ESRP1 mutations cause hearing loss, we evaluated Esrp1(-/-) mouse embryos and uncovered alterations in cochlear morphogenesis, auditory hair cell differentiation, and cell fate specification. Transcriptome analysis revealed impaired expression and splicing of genes with essential roles in cochlea development and auditory function. Aberrant splicing of Fgfr2 blocked stria vascularis formation due to erroneous ligand usage, which was corrected by reducing Fgf9 gene dosage. These findings implicate mutations in ESRP1 as a cause of SNHL and demonstrate the complex interplay between alternative splicing, inner ear development, and auditory function. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice.

    Directory of Open Access Journals (Sweden)

    Silvia Ortega-Gutiérrez

    2008-07-01

    Full Text Available Phosphoprotein phosphatase 2A (PP2A, a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A and catalytic (C subunits to which a variable regulatory subunit (B can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1. Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/- tissues, which also displayed alterations in phosphoproteome content.These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.

  18. Exposure to Endocrine Disrupters and Nuclear Receptor Gene Expression in Infertile and Fertile Women from Different Italian Areas

    Directory of Open Access Journals (Sweden)

    Cinzia La Rocca

    2014-09-01

    Full Text Available Within the PREVIENI project, infertile and fertile women were enrolled from metropolitan, urban and rural Italian areas. Blood/serum levels of several endocrine disrupters (EDs (perfluorooctane sulfonate, PFOS; perfluorooctanoic acid, PFOA; di-2-ethylhexyl-phthalate, DEHP; mono-(2-ethylhexyl-phthalate, MEHP; bisphenol A, BPA were evaluated concurrently with nuclear receptors (NRs gene expression levels (ERa, ERb, AR, AhR, PPARg, PXR in peripheral blood mononuclear cells (PBMCs. Infertile women from the metropolitan area displayed significantly higher levels of: BPA compared to fertile women (14.9 vs. 0.5 ng/mL serum; BPA and MEHP compared to infertile women from urban and rural areas; enhanced expression levels of NRs, except PPARg. Infertile women from urban and rural areas had PFOA levels significantly higher than those from metropolitan areas. Our study indicates the relevance of the living environment when investigating the exposure to EDs and the modulation of the NR panel in PBMC as a suitable biomarker of the effect, to assess the EDs impact on reproductive health.

  19. Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species.

    Science.gov (United States)

    Zhang, Bao; Liu, Chao; Wang, Yaqin; Yao, Xuan; Wang, Fang; Wu, Jiangsheng; King, Graham J; Liu, Kede

    2015-06-01

    In Brassica napus, yellow petals had a much higher content of carotenoids than white petals present in a small number of lines, with violaxanthin identified as the major carotenoid compound in yellow petals of rapeseed lines. Using positional cloning we identified a carotenoid cleavage dioxygenase 4 gene, BnaC3.CCD4, responsible for the formation of flower colour, with preferential expression in petals of white-flowered B. napus lines. Insertion of a CACTA-like transposable element 1 (TE1) into the coding region of BnaC3.CCD4 had disrupted its expression in yellow-flowered rapeseed lines. α-Ionone was identified as the major volatile apocarotenoid released from white petals but not from yellow petals. We speculate that BnaC3.CCD4 may use δ- and/or α-carotene as substrates. Four variations, including two CACTA-like TEs (alleles M1 and M4) and two insertion/deletions (INDELs, alleles M2 and M3), were identified in yellow-flowered Brassica oleracea lines. The two CACTA-like TEs were also identified in the coding region of BcaC3.CCD4 in Brassica carinata. However, the two INDELs were not detected in B. napus and B. carinata. We demonstrate that the insertions of TEs in BolC3.CCD4 predated the formation of the two allotetraploids. © 2015 The Authors New Phytologist © 2015 New Phytologist Trust.

  20. Human iPSC-Derived Cerebellar Neurons from a Patient with Ataxia-Telangiectasia Reveal Disrupted Gene Regulatory Networks

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    Sam P. Nayler

    2017-10-01

    Full Text Available Ataxia-telangiectasia (A-T is a rare genetic disorder caused by loss of function of the ataxia-telangiectasia-mutated kinase and is characterized by a predisposition to cancer, pulmonary disease, immune deficiency and progressive degeneration of the cerebellum. As animal models do not faithfully recapitulate the neurological aspects, it remains unclear whether cerebellar degeneration is a neurodevelopmental or neurodegenerative phenotype. To address the necessity for a human model, we first assessed a previously published protocol for the ability to generate cerebellar neuronal cells, finding it gave rise to a population of precursors highly enriched for markers of the early hindbrain such as EN1 and GBX2, and later more mature cerebellar markers including PTF1α, MATH1, HOXB4, ZIC3, PAX6, and TUJ1. RNA sequencing was used to classify differentiated cerebellar neurons generated from integration-free A-T and control induced pluripotent stem cells. Comparison of RNA sequencing data with datasets from the Allen Brain Atlas reveals in vitro-derived cerebellar neurons are transcriptionally similar to discrete regions of the human cerebellum, and most closely resemble the cerebellum at 22 weeks post-conception. We show that patient-derived cerebellar neurons exhibit disrupted gene regulatory networks associated with synaptic vesicle dynamics and oxidative stress, offering the first molecular insights into early cerebellar pathogenesis of ataxia-telangiectasia.

  1. The Role of Teacher Behavior Management in the Development of Disruptive Behaviors: An Intervention Study with the Good Behavior Game

    Science.gov (United States)

    Leflot, Geertje; van Lier, Pol A. C.; Onghena, Patrick; Colpin, Hilde

    2010-01-01

    The role of teacher behavior management for children's disruptive behavior development (hyperactive and oppositional behavior) was investigated using a universal classroom preventive intervention study. Five-hundred seventy children were followed from second to third grade of elementary school. Observations of teacher behavior management and…

  2. Gene expression trees in lymphoid development

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    Schliep Alexander

    2007-10-01

    Full Text Available Abstract Background The regulatory processes that govern cell proliferation and differentiation are central to developmental biology. Particularly well studied in this respect is the lymphoid system due to its importance for basic biology and for clinical applications. Gene expression measured in lymphoid cells in several distinguishable developmental stages helps in the elucidation of underlying molecular processes, which change gradually over time and lock cells in either the B cell, T cell or Natural Killer cell lineages. Large-scale analysis of these gene expression trees requires computational support for tasks ranging from visualization, querying, and finding clusters of similar genes, to answering detailed questions about the functional roles of individual genes. Results We present the first statistical framework designed to analyze gene expression data as it is collected in the course of lymphoid development through clusters of co-expressed genes and additional heterogeneous data. We introduce dependence trees for continuous variates, which model the inherent dependencies during the differentiation process naturally as gene expression trees. Several trees are combined in a mixture model to allow inference of potentially overlapping clusters of co-expressed genes. Additionally, we predict microRNA targets. Conclusion Computational results for several data sets from the lymphoid system demonstrate the relevance of our framework. We recover well-known biological facts and identify promising novel regulatory elements of genes and their functional assignments. The implementation of our method (licensed under the GPL is available at http://algorithmics.molgen.mpg.de/Supplements/ExpLym/.

  3. EDC IMPACT: Is exposure during pregnancy to acetaminophen/paracetamol disrupting female reproductive development?

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    Frederic Schrøder Arendrup

    2018-01-01

    Full Text Available Concern has been raised over chemical-induced disruption of ovary development during fetal life resulting in long-lasting consequences only manifesting themselves much later during adulthood. A growing body of evidence suggests that prenatal exposure to the mild analgesic acetaminophen/paracetamol can cause such a scenario. Therefore, in this review, we discuss three recent reports that collectively indicate that prenatal exposure in a period of 13.5 days post coitum in both rats and mouse can result in reduced female reproductive health. The combined data show that the exposure results in the reduction of primordial follicles, irregular menstrual cycle, premature absence of corpus luteum, as well as reduced fertility, resembling premature ovarian insufficiency syndrome in humans that is linked to premature menopause. This could especially affect the Western parts of the world, where the age for childbirth is continuously being increased and acetaminophen is recommended during pregnancy for pain and fever. We therefore highlight an urgent need for more studies to verify these data including both experimental and epidemiological approaches.

  4. EDC IMPACT: Is exposure during pregnancy to acetaminophen/paracetamol disrupting female reproductive development?

    Science.gov (United States)

    Arendrup, Frederic Schrøder; Mazaud-Guittot, Severine; Jégou, Bernard; Kristensen, David Møbjerg

    2018-01-01

    Concern has been raised over chemical-induced disruption of ovary development during fetal life resulting in long-lasting consequences only manifesting themselves much later during adulthood. A growing body of evidence suggests that prenatal exposure to the mild analgesic acetaminophen/paracetamol can cause such a scenario. Therefore, in this review, we discuss three recent reports that collectively indicate that prenatal exposure in a period of 13.5 days post coitum in both rats and mouse can result in reduced female reproductive health. The combined data show that the exposure results in the reduction of primordial follicles, irregular menstrual cycle, premature absence of corpus luteum, as well as reduced fertility, resembling premature ovarian insufficiency syndrome in humans that is linked to premature menopause. This could especially affect the Western parts of the world, where the age for childbirth is continuously being increased and acetaminophen is recommended during pregnancy for pain and fever. We therefore highlight an urgent need for more studies to verify these data including both experimental and epidemiological approaches. © 2018 The authors.

  5. The transformation of trust in China's alternative food networks: disruption, reconstruction, and development

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    Raymond Yu. Wang

    2015-06-01

    Full Text Available Food safety issues in China have received much scholarly attention, yet few studies systematically examined this matter through the lens of trust. More importantly, little is known about the transformation of different types of trust in the dynamic process of food production, provision, and consumption. We consider trust as an evolving interdependent relationship between different actors. We used the Beijing County Fair, a prominent ecological farmers' market in China, as an example to examine the transformation of trust in China's alternative food networks. We argue that although there has been a disruption of institutional trust among the general public since 2008 when the melamine-tainted milk scandal broke out, reconstruction of individual trust and development of organizational trust have been observed, along with the emergence and increasing popularity of alternative food networks. Based on more than six months of fieldwork on the emerging ecological agriculture sector in 13 provinces across China as well as monitoring of online discussions and posts, we analyze how various social factors - including but not limited to direct and indirect reciprocity, information, endogenous institutions, and altruism - have simultaneously contributed to the transformation of trust in China's alternative food networks. The findings not only complement current social theories of trust, but also highlight an important yet understudied phenomenon whereby informal social mechanisms have been partially substituting for formal institutions and gradually have been building trust against the backdrop of the food safety crisis in China.

  6. Catalase gene disruptant of the human pathogenic yeast Candida albicans is defective in hyphal growth, and a catalase-specific inhibitor can suppress hyphal growth of wild-type cells.

    Science.gov (United States)

    Nakagawa, Yoshiyuki

    2008-01-01

    Although the catalase gene (CAT1) disruptant of the human pathogenic yeast Candida albicans was viable under ordinary growth conditions, we previously found that it could not grow on YPD (yeast extract/peptone/dextrose) containing SDS or at higher growth temperatures. To investigate the pleiotrophic nature of the disruptant, we examined the effect of the catalase inhibitor 3-AT on the growth of wild-type strains. Surprisingly, the addition of 3-AT and SDS caused the wild-type cells to be non-viable on YPD plates. We found an additional phenotype of the catalase gene disruptant: it did not produce normal hyphae on Spider medium. Hyphal growth was observed in a CAP1 (Candida AP-1-like protein gene) disruptant, a HOG1 (high-osmolarity glycerol signaling pathway gene) disruptant, and the double CAP1/HOG1 disruptant, suggesting that the defect in hyphal formation by the catalase disruptant was independent of these genes. Addition of 3-AT and SDS to hyphae-inducing media suppressed growth of normal hyphae in the wild-type strain. The potential necessity for catalase action upon exposure to hyphae-inducing conditions was confirmed by the immediate elevation of the catalase gene message. In spite of the requirement for catalase during hyphal growth, the catalase gene disruptant was capable of forming germ tubes in medium containing serum.

  7. Bisphenol A exposure disrupts the development of the locus coeruleus-noradrenergic system in mice.

    Science.gov (United States)

    Tando, So; Itoh, Kyoko; Yaoi, Takeshi; Ogi, Hiroshi; Goto, Shoko; Mori, Miyuki; Fushiki, Shinji

    2014-12-01

    It has been reported that bisphenol A (BPA), a widespread xenoestrogen employed in the production of polycarbonate plastics, affects brain development in both humans and rodents. In the present study employing mice, we examined the effects of exposure to BPA (500 μg/kg/day) during fetal and lactational periods on the development of the locus coeruleus (LC) at the age of embryonic day 18 (E18), postnatal 3 weeks (P3W), P8W and P16W. The number of tyrosine hydroxylase-immunoreactive cells (TH-IR cells) in females exposed to BPA was decreased, compared with the control females at P3W. At P8W, the number of TH-IR cells in females exposed to BPA was significantly decreased, compared with the control females, whereas the number of TH-IR cells in males exposed to BPA was significantly increased, compared with the control males, which resulted in reversed transient sexual differences in the numbers of TH-IR cells observed in the controls at P8W. However, no significant changes were demonstrated at E18 or P16W. Next, we examined the density of the fibers containing norepinephrine transporter (NET) in the anterior cingulate cortex (ACC) and prefrontal cortex, at P3W, P8W and P16W, because NET would be beneficial in identifying the targets of the LC noradrenergic neurons. There were no significant differences shown in the density of the NET-positive fibers, between the control and the groups exposed to BPA. These results suggested that BPA might disrupt the development of physiological sexual differences in the LC-noradrenergic system in mice, although further studies are necessary to clarify the underlying mechanisms. © 2014 Japanese Society of Neuropathology.

  8. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.

    OpenAIRE

    Hube, B; Sanglard, D.; Odds, F C; Hess, D.; Monod, M; Schäfer, W; Brown, A J; Gow, N A

    1997-01-01

    Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null muta...

  9. Expression of Sox genes in tooth development

    Science.gov (United States)

    KAWASAKI, KATSUSHIGE; KAWASAKI, MAIKO; WATANABE, MOMOKO; IDRUS, ERIK; NAGAI, TAKAHIRO; OOMMEN, SHELLY; MAEDA, TAKEYASU; HAGIWARA, NOBUKO; QUE, JIANWEN; SHARPE, PAUL T.; OHAZAMA, ATSUSHI

    2017-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development. PMID:26864488

  10. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  11. Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5

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    Ichisaka Tomoko

    2006-02-01

    Full Text Available Abstract Background Embryonic stem cell-specific gene (ESG 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined. Results A Blast search of mouse genomic databases failed to locate the ESG1 gene. We identified several bacterial artificial clones containing the mouse ESG1 gene and an additional ESG1-like sequence with a similar gene structure from chromosome 9. The ESG1-like sequence contained a multiple critical mutations, indicating that it was a duplicated pseudogene. The 5' flanking region of the ESG1 gene, but not that of the pseudogene, exhibited strong enhancer and promoter activity in undifferentiated ES cells by luciferase reporter assay. To study the physiological functions of the ESG1 gene, we replaced this sequence in ES cells with a β-geo cassette by homologous recombination. Despite specific expression in early embryos and germ cells, ESG1-/- mice developed normally and were fertile. We also generated ESG1-/- ES cells both by a second independent homologous recombination and directly from blastocysts derived from heterozygous intercrosses. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells. These ES cells demonstrated normal morphology, proliferation, and differentiation. Conclusion The mouse ESG1 gene, together with a duplicated pseudogene, is located on chromosome 9. Despite its specific expression in pluripotent cells and germ cells, ESG1 is dispensable for self-renewal of ES cells and establishment of germcells.

  12. Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

    Science.gov (United States)

    Tamano, Koichi; Miura, Ai

    2016-09-01

    Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

  13. A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure.

    Science.gov (United States)

    Fong, Keith S K; Hufnagel, Robert B; Khadka, Vedbar S; Corley, Michael J; Maunakea, Alika K; Fogelgren, Ben; Ahmed, Zubair M; Lozanoff, Scott

    2016-05-01

    Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse. © 2016. Published by The Company of Biologists Ltd.

  14. Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

    Science.gov (United States)

    Xian, Jian; Aitchison, Alan; Bobrow, Linda; Corbett, Gerard; Pannell, Richard; Rabbitts, Terence; Rabbitts, Pamela

    2004-09-15

    The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.

  15. Map-Based Cloning of Genes Important for Maize Anther Development

    Science.gov (United States)

    Anaya, Y.; Walbot, V.; Nan, G.

    2012-12-01

    Map-Based cloning for maize mutant MS13 . Scientists still do not understand what decides the fate of a cell in plants. Many maize genes are important for anther development and when they are disrupted, the anthers do not shed pollen, i.e. male sterile. Since the maize genome has been fully sequenced, we conduct map-based cloning using a bulk segregant analysis strategy. Using PCR (polymerase chain reaction), we look for biomarkers that are linked to our gene of interest, Male Sterile 13 (MS13). Recombinations occur more often if the biomarkers are further away from the gene, therefore we can estimate where the gene is and design more PCR primers to get closer to our gene. Genetic and molecular analysis will help distinguish the role of key genes in setting cell fates before meiosis and for being in charge of the switch from mitosis to meiosis.

  16. Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

    Science.gov (United States)

    Wee, Sunmee; Hicks, Martin J; De, Bishnu P; Rosenberg, Jonathan B; Moreno, Amira Y; Kaminsky, Stephen M; Janda, Kim D; Crystal, Ronald G; Koob, George F

    2012-01-01

    Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>105) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited ‘extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction. PMID:21918504

  17. Overexpression of pairedless Pax6 in the retina disrupts corneal development and affects lens cell survival.

    Science.gov (United States)

    Kim, Jiha; Lauderdale, James D

    2008-01-01

    The Pax6 transcription factor is required for multiple aspects of vertebrate eye development. The Pax6 gene encodes isoforms that either contain (Pax6+PD) or lack (Pax6DeltaPD) the N-terminal paired-box DNA-binding domain, in addition to the homeodomain. Alternative promoters control the expression of Pax6+PD and Pax6DeltaPD in the eye. Using a modified bacterial artificial chromosome (BAC) transgene that specifically expresses Pax6DeltaPD, but not paired-containing Pax6, in the normal endogenous pattern, we show that overexpression of Pax6DeltaPD causes a severe microphthalmic phenotype in both wild-type and Pax6-deficient (Sey(/+)) mice in a dosage-dependent manner. The microphthalmic phenotype is due to lens degeneration during embryonic development. Lens development initiates correctly, but cells in the lens undergo apoptotic cell death between E12 and E13. Concomitantly, in these mice, changes in Bmp4, Msx1, and Wnt2b expression were observed in the mesenchymal cells of the developing cornea. To visualize Pax6DeltaPD expression, we developed a dual-reporter Pax6 BAC transgene in which EGFP and DsRed demonstrate paired-containing and pairedless transcripts, respectively. In BAC transgenic mice, DsRed is predominantly expressed in the peripheral neural retina during early eye development, but not in the developing lens or cornea. Later DsRed is strongly expressed in the developing ciliary body, but not in the iris. We suggest that the ratio of Pax6+PD and Pax6DeltaPD isoforms in the distal retina is important for both cornea and lens development, either directly by controlling transcription of necessary growth factors or indirectly by controlling development of the distal neural retina.

  18. Targeted Disruption of Guanylyl Cyclase-A/Natriuretic Peptide Receptor-A Gene Provokes Renal Fibrosis and Remodeling in Null Mutant Mice: Role of Proinflammatory Cytokines

    OpenAIRE

    Das, Subhankar; Au, Edward; Krazit, Stephen T.; Pandey, Kailash N.

    2010-01-01

    Binding of atrial and brain natriuretic peptides to guanylyl cyclase-A/natriuretic peptide receptor-A produces second messenger cGMP, which plays an important role in maintaining renal and cardiovascular homeostasis. Mice carrying a targeted disruption of the Npr1 gene coding for guanylyl cyclase-A/natriuretic peptide receptor-A exhibit changes that are similar to those that occur in untreated human hypertension, including elevated blood pressure, cardiac hypertrophy, and congestive heart fai...

  19. Digital gene expression signatures for maize development.

    Science.gov (United States)

    Eveland, Andrea L; Satoh-Nagasawa, Namiko; Goldshmidt, Alexander; Meyer, Sandra; Beatty, Mary; Sakai, Hajime; Ware, Doreen; Jackson, David

    2010-11-01

    Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

  20. Breakpoint mapping by next generation sequencing reveals causative gene disruption in patients carrying apparently balanced chromosome rearrangements with intellectual deficiency and/or congenital malformations.

    Science.gov (United States)

    Schluth-Bolard, Caroline; Labalme, Audrey; Cordier, Marie-Pierre; Till, Marianne; Nadeau, Gwenaël; Tevissen, Hélène; Lesca, Gaétan; Boutry-Kryza, Nadia; Rossignol, Sylvie; Rocas, Delphine; Dubruc, Estelle; Edery, Patrick; Sanlaville, Damien

    2013-03-01

    Apparently balanced chromosomal rearrangements (ABCR) are associated with an abnormal phenotype in 6% of cases. This may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoint. However, breakpoint cloning using conventional methods (ie, fluorescent in situ hybridisation (FISH), Southern blot) is often laborious and time consuming. In this work, we used next generation sequencing (NGS) to locate breakpoints at the molecular level in four patients with multiple congenital abnormalities and/or intellectual deficiency (MCA/ID) who were carrying ABCR (one translocation, one complex chromosomal rearrangement and two inversions), which corresponded to nine breakpoints. Genomic imbalance was previously excluded by array comparative genomic hybridisation (CGH) in all four patients. Whole genome paired-end protocol was used to identify breakpoints. The results were verified by FISH and by PCR with Sanger sequencing. We were able to map all nine breakpoints. NGS revealed an additional breakpoint due to a cryptic inversion at a breakpoint junction in one patient. Nine of 10 breakpoints occurred in repetitive elements and five genes were disrupted in their intronic sequence (TCF4, SHANK2, PPFIA1, RAB19, KCNQ1). NGS is a powerful tool allowing rapid breakpoint cloning of ABCR at the molecular level. We showed that in three out of four patients, gene disruption could account for the phenotype, allowing adapted genetic counselling and stopping unnecessary investigations. We propose that patients carrying ABCR with an abnormal phenotype should be explored systematically by NGS once a genomic imbalance has been excluded by array CGH.

  1. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection

    Science.gov (United States)

    Yang, Edward; Arvin, Ann M.

    2016-01-01

    ABSTRACT The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles

  2. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

    Science.gov (United States)

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic

  3. Sustainable Disruptions

    DEFF Research Database (Denmark)

    Friis, Silje Alberthe Kamille; Kjær, Lykke Bloch

    2016-01-01

    Since 2012 the Sustainable Disruptions (SD) project at the Laboratory for Sustainability at Design School Kolding (DK) has developed and tested a set of design thinking tools, specifically targeting the barriers to economically, socially, and environmentally sustainable business development....... The tools have been applied in practice in collaboration with 11 small and medium sized companies (SMEs). The study investigates these approaches to further understand how design thinking can contribute to sustainable transition in a business context. The study and the findings are relevant to organizations...... invested in the issue of sustainable business development, in particular the leaders and employees of SMEs, but also to design education seeking new ways to consciously handle and teach the complexity inherent in sustainable transformation. Findings indicate that the SD design thinking approach contributes...

  4. In the absence of BYPASS1-related gene function, the bps signal disrupts embryogenesis by an auxin-independent mechanism.

    Science.gov (United States)

    Lee, Dong-Keun; Van Norman, Jaimie M; Murphy, Caroline; Adhikari, Emma; Reed, Jason W; Sieburth, Leslie E

    2012-02-01

    Development is often coordinated by biologically active mobile compounds that move between cells or organs. Arabidopsis mutants with defects in the BYPASS1 (BPS1) gene overproduce an active mobile compound that moves from the root to the shoot and inhibits growth. Here, we describe two related Arabidopsis genes, BPS2 and BPS3. Analyses of single, double and triple mutants revealed that all three genes regulate production of the same mobile compound, the bps signal, with BPS1 having the largest role. The triple mutant had a severe embryo defect, including the failure to properly establish provascular tissue, the shoot meristem and the root meristem. Aberrant expression of PINFORMED1, DR5, PLETHORA1, PLETHORA2 and WUSCHEL-LIKE HOMEOBOX5 were found in heart-stage bps triple-mutant embryos. However, auxin-induced gene expression, and localization of the PIN1 auxin efflux transporter, were intact in bps1 mutants, suggesting that the primary target of the bps signal is independent of auxin response. Thus, the bps signal identifies a novel signaling pathway that regulates patterning and growth in parallel with auxin signaling, in multiple tissues and at multiple developmental stages.

  5. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Directory of Open Access Journals (Sweden)

    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

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

    Directory of Open Access Journals (Sweden)

    Keerthi Krishnan

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

  7. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  8. Gene expression analysis of flax seed development.

    Science.gov (United States)

    Venglat, Prakash; Xiang, Daoquan; Qiu, Shuqing; Stone, Sandra L; Tibiche, Chabane; Cram, Dustin; Alting-Mees, Michelle; Nowak, Jacek; Cloutier, Sylvie; Deyholos, Michael; Bekkaoui, Faouzi; Sharpe, Andrew; Wang, Edwin; Rowland, Gordon; Selvaraj, Gopalan; Datla, Raju

    2011-04-29

    Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as

  9. RNAi-mediated disruption of neuropeptide genes, nlp-3 and nlp-12, cause multiple behavioral defects in Meloidogyne incognita.

    Science.gov (United States)

    Dash, Manoranjan; Dutta, Tushar K; Phani, Victor; Papolu, Pradeep K; Shivakumara, Tagginahalli N; Rao, Uma

    2017-08-26

    Owing to the current deficiencies in chemical control options and unavailability of novel management strategies, root-knot nematode (M. incognita) infections remain widespread with significant socio-economic impacts. Helminth nervous systems are peptide-rich and appear to be putative drug targets that could be exploited by antihelmintic chemotherapy. Herein, to characterize the novel peptidergic neurotransmitters, in silico mining of M. incognita genomic and transciptomic datasets revealed the presence of 16 neuropeptide-like protein (nlp) genes with structural hallmarks of neuropeptide preproproteins; among which 13 nlps were PCR-amplified and sequenced. Two key nlp genes (Mi-nlp-3 and Mi-nlp-12) were localized to the basal bulb and tail region of nematode body via in situ hybridization assay. Mi-nlp-3 and Mi-nlp-12 were greatly expressed (in qRT-PCR assay) in the pre-parasitic juveniles and adult females, suggesting the association of these genes in host recognition, development and reproduction of M. incognita. In vitro knockdown of Mi-nlp-3 and Mi-nlp-12 via RNAi demonstrated the significant reduction in attraction and penetration of M. incognita in tomato root in Pluronic gel medium. A pronounced perturbation in development and reproduction of NLP-silenced worms was also documented in adzuki beans in CYG growth pouches. The deleterious phenotypes obtained due to NLP knockdown suggests that transgenic plants engineered to express RNA constructs targeting nlp genes may emerge as an environmentally viable option to manage nematode problems in crop plants. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival.

    Science.gov (United States)

    Al-Laaeiby, Ayat; Kershaw, Michael J; Penn, Tina J; Thornton, Christopher R

    2016-03-24

    The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H₂O₂), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H₂O₂ treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Taken together, these results show that while melanin plays a protective role in the survival of the pathogen to oxidative killing and UV radiation, melanin does not

  11. Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival

    Directory of Open Access Journals (Sweden)

    Ayat Al-Laaeiby

    2016-03-01

    Full Text Available The dematiaceous (melanised fungus Lomentospora (Scedosporium prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2, UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN-melanin biosynthetic enzymes polyketide synthase (PKS1, tetrahydroxynapthalene reductase (4HNR and scytalone dehydratase (SCD1. Infectious propagules (spores of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Taken together, these results show that while melanin plays a protective role in the survival of the pathogen to oxidative killing and UV radiation, melanin does not

  12. Gene expression in developing watermelon fruit.

    Science.gov (United States)

    Wechter, W Patrick; Levi, Amnon; Harris, Karen R; Davis, Angela R; Fei, Zhangjun; Katzir, Nurit; Giovannoni, James J; Salman-Minkov, Ayelet; Hernandez, Alvaro; Thimmapuram, Jyothi; Tadmor, Yaakov; Portnoy, Vitaly; Trebitsh, Tova

    2008-06-05

    Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded

  13. Gene expression in developing watermelon fruit

    Directory of Open Access Journals (Sweden)

    Hernandez Alvaro

    2008-06-01

    Full Text Available Abstract Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb. Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon

  14. Thyroid disruption and reduced mental development in children from an informal e-waste recycling area: A mediation analysis.

    Science.gov (United States)

    Liu, Lian; Zhang, Bo; Lin, Kun; Zhang, Yuling; Xu, Xijin; Huo, Xia

    2018-02-01

    This paper aims to evaluate the effects of thyroid disruption on the mental development of children. A total of 258 three-year-old children in Guiyu (e-waste-exposed group) and Nanao (reference group), China were examined. FT3, FT4, TSH, lead (BPb) and cadmium (BCd) in blood were determined, and cognitive and language scores of children were assessed based on the Bayley Scales of Infant Development III. Stepwise multiple regression was used to estimate the relationship between heavy metals and cognitive and language scores; mediation analysis was performed to determine whether thyroid disruption was mechanistically involved. Medians of BPb and BCd in Guiyu were higher than that of Nanao (11.30 ± 5.38 vs. 5.77 ± 2.51 μg/dL BPb; 1.22 ± 0.55 vs. 0.72 ± 0.37 μg/L BCd, both p  0.05). In contrast, Cd correlated with neither cognitive nor language scores (both p > 0.05). Results suggest exposure to heavy metal (Pb) reduces cognitive and language skills, and affects thyroid function, but fail to confirm that thyroid disruption is involved in the neurotoxicity induced by PbCd co-exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans

    DEFF Research Database (Denmark)

    Nielsen, Jakob Blæsbjerg; Nielsen, Michael Lynge; Mortensen, Uffe Hasbro

    2008-01-01

    We have developed a transiently disrupted nkuA system in Aspergillus nidulans for efficient gene targeting. The nkuA disruption was made by inserting a counter-selectable marker flanked by a direct repeat (DR) composed of nkuA sequences. In the disrupted state, the non-homologous end-joining (NHEJ...

  16. The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer.

    Science.gov (United States)

    Wichmann, Gunnar; Rosolowski, Maciej; Krohn, Knut; Kreuz, Markus; Boehm, Andreas; Reiche, Anett; Scharrer, Ulrike; Halama, Dirk; Bertolini, Julia; Bauer, Ulrike; Holzinger, Dana; Pawlita, Michael; Hess, Jochen; Engel, Christoph; Hasenclever, Dirk; Scholz, Markus; Ahnert, Peter; Kirsten, Holger; Hemprich, Alexander; Wittekind, Christian; Herbarth, Olf; Horn, Friedemann; Dietz, Andreas; Loeffler, Markus

    2015-12-15

    Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA-) are similar to HPV-negative (DNA-) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response-related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA- tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53-mutation status for patient stratification and identify associations of an immune response-related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC. © 2015 UICC.

  17. Cadmium-induced disruption in 24-h expression of clock and redox enzyme genes in rat medial basal hypothalamus. Prevention by melatonin.

    Directory of Open Access Journals (Sweden)

    Vanesa eJiménez Ortega

    2011-03-01

    Full Text Available In a previous study we reported that a low daily p.o. dose of cadmium (Cd disrupted the circadian expression of clock and redox enzyme genes in rat medial basal hypothalamus (MBH. To assess whether melatonin could counteract Cd activity, male Wistar rats (45 days of age received CdCl2 (5 ppm and melatonin (3 μg/mL or vehicle (0.015 % ethanol in drinking water. Groups of animals receiving melatonin or vehicle alone were also included. After 1 month, MBH mRNA levels were measured by real-time PCR analysis at 6 time intervals in a 24-h cycle. In control MBH Bmal1 expression peaked at early scotophase, Per1 expression at late afternoon and Per2 and Cry2 expression at mid-scotophase, whereas neither Clock nor Cry1 expression showed significant 24-h variations. This pattern was significantly disrupted (Clock, Bmal1 or changed in phase (Per1, Per2, Cry2 by CdCl2 while melatonin counteracted the changes brought about by Cd on Per1 expression only. In animals receiving melatonin alone the 24-h pattern of MBH Per2 and Cry2 expression was disrupted. CdCl2 disrupted the 24-h rhythmicity of Cu/Zn- and Mn-superoxide dismutase (SOD, nitric oxide synthase (NOS-1, NOS-2, heme oxygenase (HO-1 and HO-2 gene expression, most of the effects being counteracted by melatonin. In particular, the co-administration of melatonin and CdCl2 increased Cu/Zn-SOD gene expression and decreased that of glutathione peroxidase (GPx, glutathione reductase (GSR and HO-2. In animals receiving melatonin alone, significant increases in mean Cu/Zn and Mn-SOD gene expression, and decreases in that of GPx, GSR, NOS-1, NOS-2, HO-1 and HO-2, were found. The results indicate that the interfering effect of melatonin on the activity of a low dose of CdCl2 on MBH clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression.

  18. Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

    Science.gov (United States)

    Liu, Jessica Ai-Jia; Lai, Frank Pui-Ling; Gui, Hong-Sheng; Sham, Mai-Har; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Hui, Chi-Chung; Ngan, Elly Sau-Wai

    2015-12-01

    Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

  19. Disruption of bone development and homeostasis by trisomy in Ts65Dn Down syndrome mice.

    Science.gov (United States)

    Blazek, Joshua D; Gaddy, Anna; Meyer, Rachel; Roper, Randall J; Li, Jiliang

    2011-02-01

    Down syndrome (DS) is a genetic disorder resulting from trisomy 21 that causes cognitive impairment, low muscle tone and craniofacial alterations. Morphometric studies of the craniofacial and appendicular skeleton in individuals with DS suggest that bone development and homeostasis are affected by trisomy. The Ts65Dn mouse model has three copies of approximately half the genes found on human chromosome 21 and exhibits craniofacial skeletal and size differences similar to those observed in humans with DS. We hypothesized that Ts65Dn and euploid mice have distinct differences in bone development and homeostasis influencing both the craniofacial and appendicular skeletal phenotypes. Quantitative assessment of structural and mechanical properties of the femur in Ts65Dn and control mice at 6 and 16 weeks of age revealed significant deficiencies in trabecular and cortical bone architecture, bone mineral density, bone formation, and bone strength in trisomic bone. Furthermore, bone mineral density and dynamic dentin formation rate of the skull and incisor, respectively, were also reduced in Ts65Dn mice, demonstrating that trisomy significantly affects both the craniofacial and appendicular skeleton. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Hox Genes in Cardiovascular Development and Diseases

    Directory of Open Access Journals (Sweden)

    Marine Roux

    2016-03-01

    Full Text Available Congenital heart defects (CHD are the leading cause of death in the first year of life. Over the past 20 years, much effort has been focused on unraveling the genetic bases of CHD. In particular, studies in human genetics coupled with those of model organisms have provided valuable insights into the gene regulatory networks underlying CHD pathogenesis. Hox genes encode transcription factors that are required for the patterning of the anterior–posterior axis in the embryo. In this review, we focus on the emerging role of anteriorly expressed Hox genes (Hoxa1, Hoxb1, and Hoxa3 in cardiac development, specifically their contribution to patterning of cardiac progenitor cells and formation of the great arteries. Recent evidence regarding the cooperative regulation of heart development by Hox proteins with members of the TALE-class of homeodomain proteins such as Pbx and Meis transcription factors is also discussed. These findings are highly relevant to human pathologies as they pinpoint new genes that increase susceptibility to cardiac anomalies and provide novel mechanistic insights into CHD.

  1. Disruption of Wnt/β-catenin signaling in odontoblasts and cementoblasts arrests tooth root development in postnatal mouse teeth

    National Research Council Canada - National Science Library

    Zhang, Ran; Yang, Guan; Wu, Ximei; Xie, Jing; Yang, Xiao; Li, Tiejun

    2013-01-01

    .... In the present study, we conditionally knock out β-catenin gene (Ctnnb1) within developing odontoblasts and cementoblasts during the development of tooth roots, and observed rootless molars as well as incomplete incisors...

  2. Identification of disrupted AUTS2 and EPHA6 genes by array painting in a patient carrying a de novo balanced translocation t(3;7) with intellectual disability and neurodevelopment disorder.

    Science.gov (United States)

    Schneider, Anouck; Puechberty, Jacques; Ng, Bee Ling; Coubes, Christine; Gatinois, Vincent; Tournaire, Magali; Girard, Manon; Dumont, Bruno; Bouret, Pauline; Magnetto, Julia; Baghdadli, Amaria; Pellestor, Franck; Geneviève, David

    2015-12-01

    Intellectual disability (ID) is a frequent feature but is highly clinically and genetically heterogeneous. The establishment of the precise diagnosis in patients with ID is challenging due to this heterogeneity but crucial for genetic counseling and appropriate care for the patients. Among the etiologies of patients with ID, apparently balanced de novo rearrangements represent 0.6%. Several mechanisms explain the ID in patients with apparently balanced de novo rearrangement. Among them, disruption of a disease gene at the breakpoint, is frequently evoked. In this context, technologies recently developed are used to characterize precisely such chromosomal rearrangements. Here, we report the case of a boy with ID, facial features and autistic behavior who is carrying a de novo balanced reciprocal translocation t(3;7)(q11.2;q11.22)dn. Using microarray analysis, array painting (AP) technology combined with molecular study, we have identified the interruption of the autism susceptibility candidate 2 gene (AUTS2) and EPH receptor A6 gene (EPHA6). We consider that the disruption of AUTS2 explains the phenotype of the patient; the exact role of EPHA6 in human pathology is not well defined. Based on the observation of recurrent germinal and somatic translocations involving AUTS2 and the molecular environment content, we put forward the hypothesis that the likely chromosomal mechanism responsible for the translocation could be due either to replicative stress or to recombination-based mechanisms. © 2015 Wiley Periodicals, Inc.

  3. Disruptive firms

    OpenAIRE

    Coccia, Mario

    2017-01-01

    This study proposes the concept of disruptive firms: they are firms with market leadership that deliberate introduce new and improved generations of durable goods that destroy, directly or indirectly, similar products present in markets in order to support their competitive advantage and/or market leadership. These disruptive firms support technological and industrial change and induce consumers to buy new products to adapt to new socioeconomic environment. In particular, disruptive firms gen...

  4. Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells [Retraction

    Directory of Open Access Journals (Sweden)

    Yu L

    2017-06-01

    Full Text Available Yu L, Wang XL, Zhu D, et al. Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells. OncoTargets and Therapy. 2015;8:37–44 was published subsequent to Hu Z, Yu L, Zhu D, et al. Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. BioMed Research International. 2014 (2014, Article ID 612823.When comparing the above papers it becomes apparent that they have an unacceptably high degree of similarity and re-use. Accordingly, the Editor-in-Chief and Publisher have issued this Notice of Retraction.This retraction relates toThis retraction also relates to  

  5. Disruption of the Eng18B ENGase gene in the fungal biocontrol agent Trichoderma atroviride affects growth, conidiation and antagonistic ability.

    Directory of Open Access Journals (Sweden)

    Mukesh K Dubey

    Full Text Available The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ(8 barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous

  6. Epilepsy-causing sequence variations in SIK1 disrupt synaptic activity response gene expression and affect neuronal morphology.

    Science.gov (United States)

    Pröschel, Christoph; Hansen, Jeanne N; Ali, Adil; Tuttle, Emily; Lacagnina, Michelle; Buscaglia, Georgia; Halterman, Marc W; Paciorkowski, Alex R

    2017-02-01

    SIK1 syndrome is a newly described developmental epilepsy disorder caused by heterozygous mutations in the salt-inducible kinase SIK1. To better understand the pathophysiology of SIK1 syndrome, we studied the effects of SIK1 pathogenic sequence variations in human neurons. Primary human fetal cortical neurons were transfected with a lentiviral vector to overexpress wild-type and mutant SIK1 protein. We evaluated the transcriptional activity of known downstream gene targets in neurons expressing mutant SIK1 compared with wild type. We then assayed neuronal morphology by measuring neurite length, number and branching. Truncating SIK1 sequence variations were associated with abnormal MEF2C transcriptional activity and decreased MEF2C protein levels. Epilepsy-causing SIK1 sequence variations were associated with significantly decreased expression of ARC (activity-regulated cytoskeletal-associated) and other synaptic activity response element genes. Assay of mRNA levels for other MEF2C target genes NR4A1 (Nur77) and NRG1, found significantly, decreased the expression of these genes as well. The missense p.(Pro287Thr) SIK1 sequence variation was associated with abnormal neuronal morphology, with significant decreases in mean neurite length, mean number of neurites and a significant increase in proximal branches compared with wild type. Epilepsy-causing SIK1 sequence variations resulted in abnormalities in the MEF2C-ARC pathway of neuronal development and synapse activity response. This work provides the first insights into the mechanisms of pathogenesis in SIK1 syndrome, and extends the ARX-MEF2C pathway in the pathogenesis of developmental epilepsy.

  7. Heterochronic genes in plant evolution and development

    Directory of Open Access Journals (Sweden)

    Koen eGeuten

    2013-09-01

    Full Text Available Evolution of morphology includes evolutionary shifts of developmental processes in space or in time. Heterochronic evolution is defined as a temporal shift. The concept of heterochrony has been very rewarding to investigators of both animal and plant developmental evolution, because it has strong explanatory power when trying to understand morphological diversity. While for animals, extensive literature on heterochrony developed along with the field of evolution of development, in plants the concept has been applied less often and is less elaborately developed. Yet novel genetic findings highlight heterochrony as a developmental and evolutionary process in plants. Similar to what has been found for the worm Caenorhabditis, a heterochronic gene pathway controlling developmental timing has been elucidated in flowering plants. Two antagonistic microRNA’s miR156 and miR172 target two gene families of transcription factors, SQUAMOSA PROMOTOR BINDING PROTEIN LIKE and APETALA2-like respectively. In this review, we propose that this finding now allows the molecular investigation of cases of heterochronic evolution in plants. We illustrate this point by examining microRNA expression patterns in the Antirrhinum majus incomposita and choripetala heterochronic mutants. Some of the more beautiful putative cases of heterochronic evolution can be found outside flowering plants, but little is known about the extent of conservation of this flowering plant pathway in other land plants. We show that the expression of an APETALA2-like gene decreases with age in a fern species. This contributes to the idea that ferns share some heterochronic gene functions with flowering plants.

  8. Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

    Science.gov (United States)

    van Der Geize, R; Hessels, G I; van Gerwen, R; Vrijbloed, J W; van Der Meijden, P; Dijkhuizen, L

    2000-05-01

    Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

  9. Mutations in THAP1/DYT6 reveal that diverse dystonia genes disrupt similar neuronal pathways and functions.

    Directory of Open Access Journals (Sweden)

    Zuchra Zakirova

    2018-01-01

    Full Text Available Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in THAP1 [THAP (Thanatos-associated protein domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, THAP1 downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the in vivo effect of a heterozygote Thap1 C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia.

  10. Disruption of the Gene Encoding Endo-β-1, 4-Xylanase Affects the Growth and Virulence of Sclerotinia sclerotiorum.

    Science.gov (United States)

    Yu, Yang; Xiao, Jifen; Du, Jiao; Yang, Yuheng; Bi, Chaowei; Qing, Ling

    2016-01-01

    Sclerotinia sclerotiorum (Lib.) de Bary is a devastating fungal pathogen with worldwide distribution. S. sclerotiorum is a necrotrophic fungus that secretes many cell wall-degrading enzymes (CWDEs) that destroy plant's cell-wall components. Functional analyses of the genes that encode CWDEs will help explain the mechanisms of growth and pathogenicity of S. sclerotiorum. Here, we isolated and characterized a gene SsXyl1 that encoded an endo-β-1, 4-xylanase in S. sclerotiorum. The SsXyl1 expression showed a slight increase during the development and germination stages of sclerotia and a dramatic increase during infection. The expression of SsXyl1 was induced by xylan. The SsXyl1 deletion strains produce aberrant sclerotia that could not germinate to form apothecia. The SsXyl1 deletion strains also lost virulence to the hosts. This study demonstrates the important roles of endo-β-1, 4-xylanase in the growth and virulence of S. sclerotiorum.

  11. Gene network disruptions and neurogenesis defects in the adult Ts1Cje mouse model of Down syndrome.

    Directory of Open Access Journals (Sweden)

    Chelsee A Hewitt

    Full Text Available BACKGROUND: Down syndrome (DS individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease. METHODOLOGY/PRINCIPAL FINDINGS: To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased. CONCLUSIONS/SIGNIFICANCE: We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals.

  12. Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

    Science.gov (United States)

    Chernova, Tatyana; Murphy, Fiona A; Galavotti, Sara; Sun, Xiao-Ming; Powley, Ian R; Grosso, Stefano; Schinwald, Anja; Zacarias-Cabeza, Joaquin; Dudek, Kate M; Dinsdale, David; Le Quesne, John; Bennett, Jonathan; Nakas, Apostolos; Greaves, Peter; Poland, Craig A; Donaldson, Ken; Bushell, Martin; Willis, Anne E; MacFarlane, Marion

    2017-11-06

    Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Gene networks controlling Arabidopsis thaliana flower development.

    Science.gov (United States)

    Ó'Maoiléidigh, Diarmuid Seosamh; Graciet, Emmanuelle; Wellmer, Frank

    2014-01-01

    The formation of flowers is one of the main models for studying the regulatory mechanisms that underlie plant development and evolution. Over the past three decades, extensive genetic and molecular analyses have led to the identification of a large number of key floral regulators and to detailed insights into how they control flower morphogenesis. In recent years, genome-wide approaches have been applied to obtaining a global view of the gene regulatory networks underlying flower formation. Furthermore, mathematical models have been developed that can simulate certain aspects of this process and drive further experimentation. Here, we review some of the main findings made in the field of Arabidopsis thaliana flower development, with an emphasis on recent advances. In particular, we discuss the activities of the floral organ identity factors, which are pivotal for the specification of the different types of floral organs, and explore the experimental avenues that may elucidate the molecular mechanisms and gene expression programs through which these master regulators of flower development act. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  14. Evidence of gene-environment correlation for peer difficulties: disruptive behaviors predict early peer relation difficulties in school through genetic effects.

    Science.gov (United States)

    Boivin, Michel; Brendgen, Mara; Vitaro, Frank; Forget-Dubois, Nadine; Feng, Bei; Tremblay, Richard E; Dionne, Ginette

    2013-02-01

    Early disruptive behaviors, such as aggressive and hyperactive behaviors, known to be influenced by genetic factors, have been found to predict early school peer relation difficulties, such as peer rejection and victimization. However, there is no consensus regarding the developmental processes underlying this predictive association. Genetically informative designs, such as twin studies, are well suited for investigating the underlying genetic and environmental etiology of this association. The main goal of the present study was to examine the possible establishment of an emerging gene-environment correlation linking disruptive behaviors to peer relationship difficulties during the first years of school. Participants were drawn from an ongoing longitudinal study of twins who were assessed with respect to their social behaviors and their peer relation difficulties in kindergarten and in Grade 1 through peer nominations measures and teacher ratings. As predicted, disruptive behaviors were concurrently and predictively associated with peer relation difficulties. Multivariate analyses of these associations indicate that they were mainly accounted for by genetic factors. These results emphasize the need to adopt an early and persistent prevention framework targeting both the child and the peer context to alleviate the establishment of a negative coercive process and its consequences.

  15. Streptomyces venezuelae ISP5230 Maintains Excretion of Jadomycin upon Disruption of the MFS Transporter JadL Located within the Natural Product Biosynthetic Gene Cluster.

    Science.gov (United States)

    Forget, Stephanie M; McVey, Jennifer; Vining, Leo C; Jakeman, David L

    2017-01-01

    JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.

  16. Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae).

    Science.gov (United States)

    Zhang, Rui; Guo, Chunce; Zhang, Wengen; Wang, Peipei; Li, Lin; Duan, Xiaoshan; Du, Qinggao; Zhao, Liang; Shan, Hongyan; Hodges, Scott A; Kramer, Elena M; Ren, Yi; Kong, Hongzhi

    2013-03-26

    Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.

  17. Improving the Efficiency of Homologous Gene Replacement by Disrupting the NHEJ Pathway for Gene KusA in the Oleaginous Fungus Mortierella alpina

    Science.gov (United States)

    Krueger, Kathleen; Dai, Ziyu; Uzuner, Uger

    2012-11-01

    Mortierella alpina, a oleaginous filamentous fungus, is one of industrial fungal strains known for the production of arachidonic acid. It is also of particular interest for hydrocarbon biofuel production since it is able to produce up to 50% of its mass in rich, long-chain polyunsaturated fatty acids [PUFA's]. In addition to high fatty acid production, M. alpina like many other oleaginous fungi, already have mechanisms for accumulating significant concentrations of hydrophobic compounds making it a naturally equipped candidate to handle potential toxic concentrations of hydrocarbons. The goal of this study was to develop an efficient transformation method for this strain, hence allowing researchers to further manipulate these fungi for further improvement of lipid production. Included was optimization of best culture medium for growth and maintenance, optimal conditions for protoplast generation, and replacement of the homologous KusA gene. A successful deletion of KusA gene within biotechnologically important M. alpina could enable homologous recombination of other genes of interest in a higher frequency. This capacity may also improve the advancing the production of microbial oils for bioenergy and arachidonic acid human health applications.

  18. Disruption of a rice gene for α-glucan water dikinase, OsGWD1, leads to hyperaccumulation of starch in leaves but exhibits limited effects on growth

    Directory of Open Access Journals (Sweden)

    Tatsuro eHirose

    2013-05-01

    Full Text Available To identify potential regulators of photoassimilate partitioning, we screened for rice mutant plants that accumulate high levels of starch in the leaf blades, and a mutant line Leaf Starch Excess 1 (LSE1 was obtained and characterized. The starch content in the leaf blades of LSE1 was more than 10-fold higher than that in wild-type plants throughout the day, while the sucrose content was unaffected. The gene responsible for the LSE1 phenotype was identified by gene mapping to be a gene encoding α-glucan water dikinase, OsGWD1 (Os06g0498400, and a 3.4-kbp deletion of the gene was found in the mutant plant. Despite the hyperaccumulation of starch in their leaf blades, LSE1 plants exhibited no significant change in vegetative growth, presenting a clear contrast to the reported mutants of Arabidopsis thaliana and Lotus japonicus in which disruption of the genes for α-glucan water dikinase leads to marked inhibition of vegetative growth. In reproductive growth, however, LSE1 exhibited fewer panicles per plant, lower percentage of ripened grains and smaller grains; consequently, the grain yield was lower in LSE1 plants than in wild-type plants by 20~40 %. Collectively, although α-glucan water dikinase was suggested to have universal importance in leaf starch degradation in higher plants, the physiological priority of leaf starch in photoassimilate allocation may vary among plant species.

  19. Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

    Science.gov (United States)

    Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

    2016-09-01

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

  20. A Behavioral Framework for Managing Massive Airline Flight Disruptions through Crisis Management, Organization Development, and Organization Learning

    Science.gov (United States)

    Larsen, Tulinda Deegan

    In this study the researcher provides a behavioral framework for managing massive airline flight disruptions (MAFD) in the United States. Under conditions of MAFD, multiple flights are disrupted throughout the airline's route network, customer service is negatively affected, additional costs are created for airlines, and governments intervene. This study is different from other studies relating to MAFD that have focused on the operational, technical, economic, financial, and customer service impacts. The researcher argues that airlines could improve the management of events that led to MAFD by applying the principles of crisis management where the entire organization is mobilized, rather than one department, adapting organization development (OD) interventions to implement change and organization learning (OL) processes to create culture of innovation, resulting in sustainable improvement in customer service, cost reductions, and mitigation of government intervention. At the intersection of crisis management, OD, and OL, the researcher has developed a new conceptual framework that enhances the resiliency of individuals and organizations in responding to unexpected-yet-recurring crises (e.g., MAFD) that impact operations. The researcher has adapted and augmented Lalonde's framework for managing crises through OD interventions by including OL processes. The OD interventions, coupled with OL, provide a framework for airline leaders to manage more effectively events that result in MAFD with the goal of improving passenger satisfaction, reducing costs, and preventing further government intervention. Further research is warranted to apply this conceptual framework to unexpected-yet-recurring crises that affect operations in other industries.

  1. Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons.

    Science.gov (United States)

    Powrozek, Teresa A; Olson, Eric C

    2012-11-01

    Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Disruptive Mood Dysregulation Disorder

    Science.gov (United States)

    ... conduct research on numerous areas of study, including cognition, genetics, epidemiology, and psychiatry. The studies take place ... Leibenluft, Senior Investigator and Chief of the NIMH Emotion and Development Branch, explains the history of Disruptive ...

  3. Digital Disruption

    DEFF Research Database (Denmark)

    Rosenstand, Claus Andreas Foss

    det digitale domæne ud over det niveau, der kendetegner den nuværende debat, så præsenteres der ny viden om digital disruption. Som noget nyt udlægges Clayton Christens teori om disruptiv innovation med et særligt fokus på små organisationers mulighed for eksponentiel vækst. Specielt udfoldes...... forholdet mellem disruption og den stadig accelererende digitale udvikling i konturerne til ny teoridannelse om digital disruption. Bogens undertitel ”faretruende og fascinerende forandringer” peger på, at der er behov for en nuanceret debat om digital disruption i modsætning til den tone, der er slået an i...... videre kalder et ”disruption-råd”. Faktisk er rådet skrevet ind i 2016 regeringsgrundlaget for VLK-regeringen. Disruption af organisationer er ikke et nyt fænomen; men hastigheden, hvormed det sker, er stadig accelererende. Årsagen er den globale mega-trend: Digitalisering. Og derfor er specielt digital...

  4. Future Scenario Development from Disruptive Exploration Technologies and Business Models in the U.S. Geothermal Industry

    Energy Technology Data Exchange (ETDEWEB)

    Wall, Anna

    2017-05-01

    With recent trends toward intermittent renewable energy sources in the U.S., the geothermal industry in its current form faces a crossroad: adapt, disrupt, or be left behind. Strategic planning with scenario analysis offers a framework to characterize plausible views of the future given current trends - as well as disruptions to the status quo. To inform strategic planning in the Department of Energy's (DOE) Geothermal Technology Office (GTO), the Geothermal Vision Study is tasked with offering data-driven pathways for future geothermal development. Scenario analysis is a commonly used tool in private industry corporate strategic planning as a way to prioritize and manage large investments in light of uncertainty and risk. Since much of the uncertainty and risk in a geothermal project is believed to occur within early stage exploration and drilling, this paper focuses on the levers (technical and financial) within the exploration process that can be pulled to affect change. Given these potential changes, this work first qualitatively explores potential shifts to the geothermal industry. Future work within the Geothermal Vision Study will incorporate these qualitative scenarios quantitatively, in competition with other renewable and conventional energy industries.

  5. Miswiring the brain: Δ9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway

    Science.gov (United States)

    Tortoriello, Giuseppe; Morris, Claudia V; Alpar, Alan; Fuzik, Janos; Shirran, Sally L; Calvigioni, Daniela; Keimpema, Erik; Botting, Catherine H; Reinecke, Kirstin; Herdegen, Thomas; Courtney, Michael; Hurd, Yasmin L; Harkany, Tibor

    2014-01-01

    Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ9-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring. PMID:24469251

  6. Disrupting Na+,HCO3--cotransporter NBCn1 (Slc4a7) delays murine breast cancer development

    DEFF Research Database (Denmark)

    Lee, S.; Axelsen, T. V.; Andersen, Anne Poder

    2016-01-01

    . Genome-wide association studies have indicated a possible link between the Na(+),HCO3(-)-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis...... and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT......) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently...

  7. Production and clinical development of nanoparticles for gene delivery

    Directory of Open Access Journals (Sweden)

    Jie Chen

    2016-01-01

    Full Text Available Gene therapy is a promising strategy for specific treatment of numerous gene-associated human diseases by intentionally altering the gene expression in pathological cells. A successful clinical application of gene-based therapy depends on an efficient gene delivery system. Many efforts have been attempted to improve the safety and efficiency of gene-based therapies. Nanoparticles have been proved to be the most promising vehicles for clinical gene therapy due to their tunable size, shape, surface, and biological behaviors. In this review, the clinical development of nanoparticles for gene delivery will be particularly highlighted. Several promising candidates, which are closest to clinical applications, will be briefly reviewed. Then, the recent developments of nanoparticles for clinical gene therapy will be identified and summarized. Finally, the development of nanoparticles for clinical gene delivery in future will be prospected.

  8. Translocations of 11q13 in mantle cell lymphoma fail to disrupt the S mu bp-2 gene.

    Science.gov (United States)

    Gulley, M L; Zhang, Q; Gascoyne, R D; DuPont, B R; Banks, P M; Cho, C G; Huang, J M; Montalvo, E A

    We recently cloned a gene whose protein product binds to the Epstein-Barr virus BZLF1 gene promoter. The same gene has been previously cloned by another group who named it S mu bp-2 because its protein product binds to the S mu motif of the immunoglobulin heavy chain gene where it is postulated to function in immunoglobulin class switching. In the current study, we confirm that the S mu bp-2 gene is located on chromosome 11q13, a locus known to be altered by translocation in 50-70% of mantle cell lymphomas. We used Southern blot analysis to determine whether the S mu bp-2 gene was structurally rearranged in any of 25 mantle cell lymphomas. We found no evidence of rearrangement in any of these lymphomas including 18 that were proven to contain t(11;14) by cytogenetic analysis. These data suggest that structural alteration of the S mu bp-2 gene is not an underlying mechanism of tumorigenesis in mantle cell lymphomas.

  9. Expanding the test set: Chemicals with potential to disrupt mammalian brain development

    Science.gov (United States)

    High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxcants. As new assays are developed, a "training set' of chemicals i...

  10. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    Science.gov (United States)

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. Copyright © 2015 Elsevier GmbH. All rights reserved.

  11. Disrupting Business

    DEFF Research Database (Denmark)

    Cox, Geoff; Bazzichelli, Tatiana

    Disruptive Business explores some of the interconnections between art, activism and the business concept of disruptive innovation. With a backdrop of the crisis of financial capitalism, austerity cuts in the cultural sphere, the idea is to focus on potential art strategies in relation to a broken...... economy. In a perverse way, we ask whether this presents new opportunities for cultural producers to achieve more autonomy over their production process. If it is indeed possible, or desirable, what alternative business models emerge? The book is concerned broadly with business as material for reinvention...

  12. Development of SPR Immunosensing System Using Microchannel Cell for Simultaneous Detection of Several Endocrine-Disrupting Chemicals

    Science.gov (United States)

    Miura, Norio; K. Vengatajalabathy, Gobi; Shoyama, Yukihiro; Maeda, Hideaki; Kawazumi, Hirofumi; Iwasaka, Hiroyuki

    The endocrine-disrupting chemicals affect the functioning of hormones of animals and are called “environmental hormones". They exist in various environments at very low concentrations in the range of ppt to ppb levels. Thus, highly sensitive determination of environmental hormones with high selectivity is indispensable in regulating the ecosystem, and the instrumentation that allows the on-site analysis of environmental hormones is paid much attention. In this research project, we are aiming at development of new optical immunosensing system for highly sensitive, selective, on-site and simultaneous detection of several environmental hormones at low cost. We report here the results of our research investigations on application of immunosensing technique to highly sensitive detection of environmental hormones, preparation of monoclonal antibodies, fabrication of the microchannel, miniaturization of the surface-plasmon-resonance detector, design of the compact total-sensing-system.

  13. Intestinal microbiome disruption in patients in a long-term acute care hospital: A case for development of microbiome disruption indices to improve infection prevention.

    Science.gov (United States)

    Halpin, Alison Laufer; de Man, Tom J B; Kraft, Colleen S; Perry, K Allison; Chan, Austin W; Lieu, Sung; Mikell, Jeffrey; Limbago, Brandi M; McDonald, L Clifford

    2016-07-01

    Composition and diversity of intestinal microbial communities (microbiota) are generally accepted as a risk factor for poor outcomes; however, we cannot yet use this information to prevent adverse outcomes. Stool was collected from 8 long-term acute care hospital patients experiencing diarrhea and 2 fecal microbiota transplant donors; 16S rDNA V1-V2 hypervariable regions were sequenced. Composition and diversity of each sample were described. Stool was also tested for Clostridium difficile, vancomycin-resistant enterococci (VRE), and carbapenem-resistant Enterobacteriaceae. Associations between microbiota diversity and demographic and clinical characteristics, including antibiotic use, were analyzed. Antibiotic exposure and Charlson Comorbidity Index were inversely correlated with diversity (Spearman = -0.7). Two patients were positive for VRE; both had microbiomes dominated by Enterococcus faecium, accounting for 67%-84% of their microbiome. Antibiotic exposure correlated with diversity; however, other environmental and host factors not easily obtainable in a clinical setting are also known to impact the microbiota. Therefore, direct measurement of microbiome disruption by sequencing, rather than reliance on surrogate markers, might be most predictive of adverse outcomes. If and when microbiome characterization becomes a standard diagnostic test, improving our understanding of microbiome dynamics will allow for interpretation of results to improve patient outcomes. Published by Elsevier Inc.

  14. Methylation at 3'LCR of HPV16 can be affected by patient age and disruption of E1 or E2 genes.

    Science.gov (United States)

    Filho, Sérgio Menezes Amaro; Bertoni, Neilane; Brant, Ayslan Castro; Vidal, João Paulo Castello Branco; Felix, Shayany Pinto; Cavalcanti, Silvia Maria Baeta; Carestiato, Fernanda N; Martins, Luís Felipe Leite; Almeida, Liz Maria de; Moreira, Miguel Angelo Martins

    2017-03-15

    CpG methylation at early promoter of HPV16 DNA, in the 3' end of the Long Control Region (3'LCR), has been associated to the presence of episomal forms of viral genome and, consequently, intact E1 and E2 ORFs. The DNA methylation would block the access of E2 viral protein to the E2 binding sites at early-promoter. However, is still unclear if methylation at 3'LCR of HPV16 DNA can also vary depending of other tumor characteristics in addition to viral DNA physical state. In this study, we evaluate whether the methylation level at the five CpG located at 3'LCR of HPV16 is associated to patient age and E1 and/or E2 ORFs integrity. DNA pyrosequencing was used to measure the methylation level in 69 invasive cervical cancer samples obtained from biopsies of patients attended at Brazilian National Institute of Cancer (INCA). PCR amplifications were performed to assess disruption status of E1 and E2 genes of HPV16. The methylation average per sample ranged widely, from frames was associated with high levels of DNA methylation, and older patients showed higher levels of methylation than younger ones independently of viral genome disruption. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Interpersonal Relatedness and Self-Definition in Normal and Disrupted Personality Development: Retrospect and Prospect

    Science.gov (United States)

    Luyten, Patrick; Blatt, Sidney J.

    2013-01-01

    Two-polarities models of personality propose that personality development evolves through a dialectic synergistic interaction between two fundamental developmental psychological processes across the life span--the development of interpersonal relatedness on the one hand and of self-definition on the other. This article offers a broad review of…

  16. Transcription of the soybean leghemoglobin genes during nodule development

    DEFF Research Database (Denmark)

    Marcker, Anne; Ø Jensen, Erik; Marcker, Kjeld A

    1984-01-01

    During the early stages of soybean nodule development the leghemoglobin (Lb) genes are activated sequentially in the opposite order to which they are arranged in the soybean genome. At a specific stage after the initial activation of all the Lb genes, a large increment occurs in the transcription...... of the Lb(c1), Lb(c3) and Lb(a) genes while the transcription of the Lb(c2) gene is not amplified to a similar extent. All the Lb genes retain significant activity for a long period during the lifetime of a nodule. Consequently the soybean Lb genes are not regulated by a developmental gene switching...

  17. Control of Plant Trichome Development by a Cotton Fiber MYB GeneW⃞

    Science.gov (United States)

    Wang, Shui; Wang, Jia-Wei; Yu, Nan; Li, Chun-Hong; Luo, Bin; Gou, Jin-Ying; Wang, Ling-Jian; Chen, Xiao-Ya

    2004-01-01

    Cotton (Gossypium spp) plants produce seed trichomes (cotton fibers) that are an important commodity worldwide; however, genes controlling cotton fiber development have not been characterized. In Arabidopsis thaliana the MYB gene GLABRA1 (GL1) is a central regulator of trichome development. Here, we show that promoter of a cotton fiber gene, RD22-like1 (RDL1), contains a homeodomain binding L1 box and a MYB binding motif that confer trichome-specific expression in Arabidopsis. A cotton MYB protein GaMYB2/Fiber Factor 1 transactivated the RDL1 promoter both in yeast and in planta. Real-time PCR and in situ analysis showed that GaMYB2 is predominantly expressed early in developing cotton fibers. After transferring into Arabidopsis, GL1∷GaMYB2 rescued trichome formation of a gl1 mutant, and interestingly, 35S∷GaMYB2 induced seed-trichome production. We further demonstrate that the first intron of both GL1 and GaMYB2 plays a role in patterning trichomes: it acts as an enhancer in trichome and a repressor in nontrichome cells, generating a trichome-specific pattern of MYB gene expression. Disruption of a MYB motif conserved in intron 1 of GL1, WEREWOLF, and GaMYB2 genes affected trichome production. These results suggest that cotton and Arabidopsis use similar transcription factors for regulating trichomes and that GaMYB2 may be a key regulator of cotton fiber development. PMID:15316114

  18. Control of plant trichome development by a cotton fiber MYB gene.

    Science.gov (United States)

    Wang, Shui; Wang, Jia-Wei; Yu, Nan; Li, Chun-Hong; Luo, Bin; Gou, Jin-Ying; Wang, Ling-Jian; Chen, Xiao-Ya

    2004-09-01

    Cotton (Gossypium spp) plants produce seed trichomes (cotton fibers) that are an important commodity worldwide; however, genes controlling cotton fiber development have not been characterized. In Arabidopsis thaliana the MYB gene GLABRA1 (GL1) is a central regulator of trichome development. Here, we show that promoter of a cotton fiber gene, RD22-like1 (RDL1), contains a homeodomain binding L1 box and a MYB binding motif that confer trichome-specific expression in Arabidopsis. A cotton MYB protein GaMYB2/Fiber Factor 1 transactivated the RDL1 promoter both in yeast and in planta. Real-time PCR and in situ analysis showed that GaMYB2 is predominantly expressed early in developing cotton fibers. After transferring into Arabidopsis, GL1::GaMYB2 rescued trichome formation of a gl1 mutant, and interestingly, 35S::GaMYB2 induced seed-trichome production. We further demonstrate that the first intron of both GL1 and GaMYB2 plays a role in patterning trichomes: it acts as an enhancer in trichome and a repressor in nontrichome cells, generating a trichome-specific pattern of MYB gene expression. Disruption of a MYB motif conserved in intron 1 of GL1, WEREWOLF, and GaMYB2 genes affected trichome production. These results suggest that cotton and Arabidopsis use similar transcription factors for regulating trichomes and that GaMYB2 may be a key regulator of cotton fiber development.

  19. Impact of Low-Level Thyroid Hormone Disruption Induced by Propylthiouracil on Brain Development and Function.*

    Science.gov (United States)

    The critical role of thyroid hormone (TH) in brain development is well established, severe deficiencies leading to significant neurological dysfunction. Much less information is available on more modest perturbations of TH on brain function. The present study induced varying degr...

  20. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.

    Science.gov (United States)

    Fang, Yufeng; Tyler, Brett M

    2016-01-01

    Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9-induced DNA double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end-joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology-directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR-mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen. © 2015 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY JOHN WILEY & SONS LTD AND BSPP.

  1. Evidence for association between Disrupted-in-schizophrenia 1 (DISC1 gene polymorphisms and autism in Chinese Han population: a family-based association study

    Directory of Open Access Journals (Sweden)

    Ruan Yan

    2011-05-01

    Full Text Available Abstract Background Disrupted-in-Schizophrenia 1 (DISC1 gene is one of the most promising candidate genes for major mental disorders. In a previous study, a Finnish group demonstrated that DISC1 polymorphisms were associated with autism and Asperger syndrome. However, the results were not replicated in Korean population. To determine whether DISC1 is associated with autism in Chinese Han population, we performed a family-based association study between DISC1 polymorphisms and autism. Methods We genotyped seven tag single nucleotide polymorphisms (SNPs in DISC1, spanning 338 kb, in 367 autism trios (singleton and their biological parents including 1,101 individuals. Single SNP association and haplotype association analysis were performed using the family-based association test (FBAT and Haploview software. Results We found three SNPs showed significant associations with autism (rs4366301: G > C, Z = 2.872, p = 0.004; rs11585959: T > C, Z = 2.199, p = 0.028; rs6668845: A > G, Z = 2.326, p = 0.02. After the Bonferroni correction, SNP rs4366301, which located in the first intron of DISC1, remained significant. When haplotype were constructed with two-markers, three haplotypes displayed significant association with autism. These results were still significant after using the permutation method to obtain empirical p values. Conclusions Our study provided evidence that the DISC1 may be the susceptibility gene of autism. It suggested DISC1 might play a role in the pathogenesis of autism.

  2. Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Amos O. Abolaji

    2015-08-01

    Full Text Available The compounds 4-vinylcyclohexene 1,2-monoepoxide (VCM and 4-Vinylcyclohexene diepoxide (VCD are the two downstream metabolites of 4-vinylcyclohexene (VCH, an ovotoxic agent in mammals. In addition, VCM and VCD may be found as by-products of VCH oxidation in the environment. Recently, we reported the involvement of oxidative stress in the toxicity of VCH in Drosophila melanogaster. However, it was not possible to determine the individual contributions of VCM and VCD in VCH toxicity. Hence, we investigated the toxicity of VCM and VCD (10–1000 µM in flies after 5 days of exposure via the diet. Our results indicated impairments in climbing behaviour and disruptions in antioxidant balance and redox status evidenced by an increase in DCFH oxidation, decreases in total thiol content and glutathione-S-transferase (GST activity in the flies exposed to VCM and VCD (p<0.05. These effects were accompanied by disruptions in the transcription of the genes encoding the proteins superoxide dismutase (SOD1, kelch-like erythroid-derived cap-n-collar (CNC homology (ECH-associated protein 1 (Keap-1, mitogen activated protein kinase 2 (MAPK-2, catalase, Cyp18a1, JAFRAC 1 (thioredoxin peroxidase 1 and thioredoxin reductase 1 (TrxR-1 (p<0.05. VCM and VCD inhibited acetylcholinesterase (AChE and delta aminolevulinic acid dehydratase (δ-ALA D activities in the flies (p<0.05. Indeed, here, we demonstrated that different target enzymes and genes were modified by the electrophiles VCM and VCD in the flies. Thus, D. melanogaster has provided further lessons on the toxicity of VCM and VCD which suggest that the reported toxicity of VCH may be mediated by its transformation to VCM and VCD.

  3. Mouse model of N-acetylgalactosamine-6-sulfate sulfatase deficiency (Galns-/-) produced by targeted disruption of the gene defective in Morquio A disease.

    Science.gov (United States)

    Tomatsu, Shunji; Orii, Koji O; Vogler, Carole; Nakayama, Jun; Levy, Beth; Grubb, Jeffrey H; Gutierrez, Monica A; Shim, Soomin; Yamaguchi, Seiji; Nishioka, Tatsuo; Montano, Adriana Maria; Noguchi, Akihiko; Orii, Tadao; Kondo, Naomi; Sly, William S

    2003-12-15

    Mucopolysaccharidosis IVA is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), a lysosomal enzyme required for the stepwise degradation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). To generate a model for studies of the pathophysiology and of potential therapies, we disrupted exon 2 of Galns, the homologous murine gene. Homozygous Galns-/- mice have no detectable GALNS enzyme activity and show increased urinary glycosaminoglycan (GAGs) levels. These mice accumulate GAGs in multiple tissues including liver, kidney, spleen, heart, brain and bone marrow. At 2 months old, lysosomal storage is present primarily within reticuloendothelial cells such as Kupffer cells and cells of the sinusoidal lining of the spleen. Additionally, by 12 months old, vacuolar change is observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. In the brain, hippocampal and neocortical neurons and meningeal cells had lysosomal storage. KS and C6S were more abundant in the cytoplasm of corneal epithelial cells of Galns-/- mice compared with wild-type mice by immunohistochemistry. Radiographs revealed no change in the skeletal bones of mice up to 12 months old. Thus, targeted disruption of the murine Galns gene has produced a murine model, which shows visceral storage of GAGs but lacks the skeletal features. The complete absence of GALNS in mutant mice makes them useful for studies of pharmacokinetics and tissue targeting of recombinant GALNS designed for enzyme replacement.

  4. Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production

    Science.gov (United States)

    Nian-Qing. Shi; Brian. Davis; Fred. Sherman; Jose. Cruz; Thomas W. Jeffries

    1999-01-01

    The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization...

  5. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Engle, S.J.; Chen, J.; Tischfield, J.A. [Indiana Univ., School of Medicine, Indianapolis, IN (United States)] [and others

    1994-09-01

    Adenine phosphoribosyltransferase (APRT: EC 2.4.2.7), a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficient animals.

  6. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    Science.gov (United States)

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  7. Disruption of the sigS gene attenuates the local innate immune response to Staphylococcus aureus in a mouse mastitis model.

    Science.gov (United States)

    Peton, Vincent; Breyne, Koen; Rault, Lucie; Demeyere, Kristel; Berkova, Nadia; Meyer, Evelyne; Even, Sergine; Le Loir, Yves

    2016-04-15

    Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes. Copyright

  8. The Role of Friends' Disruptive Behavior in the Development of Children's Tobacco Experimentation: Results from a Preventive Intervention Study

    Science.gov (United States)

    van Lier, Pol A. C.; Huizink, Anja; Vuijk, Patricia

    2011-01-01

    Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends' disruptive behavior as a mediator of the effects of a classroom based intervention on children's tobacco experimentation. 433 Children (52% males) were randomly assigned to the Good…

  9. The role of friends' disruptive behavior in the development of children's tobacco experimentation: results from a preventive intervention study

    NARCIS (Netherlands)

    Van Lier, P.A.C.; Huizink, A.; Vuijk, P.

    2011-01-01

    Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends’ disruptive behavior as a mediator of the effects of a classroom based intervention on children’s tobacco experimentation. 433 Children (52%

  10. Disregard for Rules: The Early Development and Predictors of a Specific Dimension of Disruptive Behavior Disorders

    Science.gov (United States)

    Petitclerc, Amelie; Boivin, Michel; Dionne, Ginette; Zoccolillo, Mark; Tremblay, Richard E.

    2009-01-01

    Background: Disregard for rules, an important dimension of oppositional defiant and conduct disorders, is frequent during early childhood, but the development of its chronic form has not been studied during this key socialization period. This study aimed to describe the developmental trajectories of disregard for rules during early childhood and…

  11. Disruption of Transient Serotonin Accumulation by Non-Serotonin-Producing Neurons Impairs Cortical Map Development

    Directory of Open Access Journals (Sweden)

    Xiaoning Chen

    2015-01-01

    Full Text Available Polymorphisms that alter serotonin transporter SERT expression and functionality increase the risks for autism and psychiatric traits. Here, we investigate how SERT controls serotonin signaling in developing CNS in mice. SERT is transiently expressed in specific sets of glutamatergic neurons and uptakes extrasynaptic serotonin during perinatal CNS development. We show that SERT expression in glutamatergic thalamocortical axons (TCAs dictates sensory map architecture. Knockout of SERT in TCAs causes lasting alterations in TCA patterning, spatial organizations of cortical neurons, and dendritic arborization in sensory cortex. Pharmacological reduction of serotonin synthesis during the first postnatal week rescues sensory maps in SERTGluΔ mice. Furthermore, knockdown of SERT expression in serotonin-producing neurons does not impair barrel maps. We propose that spatiotemporal SERT expression in non-serotonin-producing neurons represents a determinant in early life genetic programming of cortical circuits. Perturbing this SERT function could be involved in the origin of sensory and cognitive deficits associated with neurodevelopmental disorders.

  12. A Covert Disruptive Technology: Test and Development of the Corona Satellite

    Science.gov (United States)

    Peebles, Curtis

    2008-01-01

    The launching by the Soviet Union of the Sputnik satellite in 19457 was an impetuous to the United States. The Intercontinental ballistic Missile (ICBM) that launched the Earth's first satellite, could have been armed with a nuclear warhead, that could destroy an American city. The primary intelligence requirement that the US had was to determine the actual size of the Soviet missile program. To this end, a covert, high-risk photoreconnaissance satellite was developed. The code name of this program was "Corona." This article describes the trials and eventual successes of the Corona program.

  13. Drug-Induced Apoptosis: Mechanism by which Alcohol and Many Other Drugs Can Disrupt Brain Development

    Directory of Open Access Journals (Sweden)

    John W. Olney

    2013-07-01

    Full Text Available Maternal ingestion of alcohol during pregnancy can cause a disability syndrome termed Fetal Alcohol Spectrum Disorder (FASD, which may include craniofacial malformations, structural pathology in the brain, and a variety of long-term neuropsychiatric disturbances. There is compelling evidence that exposure to alcohol during early embryogenesis (4th week of gestation can cause excessive death of cell populations that are essential for normal development of the face and brain. While this can explain craniofacial malformations and certain structural brain anomalies that sometimes accompany FASD, in many cases these features are absent, and the FASD syndrome manifests primarily as neurobehavioral disorders. It is not clear from the literature how alcohol causes these latter manifestations. In this review we will describe a growing body of evidence documenting that alcohol triggers widespread apoptotic death of neurons and oligodendroglia (OLs in the developing brain when administered to animals, including non-human primates, during a period equivalent to the human third trimester of gestation. This cell death reaction is associated with brain changes, including overall or regional reductions in brain mass, and long-term neurobehavioral disturbances. We will also review evidence that many drugs used in pediatric and obstetric medicine, including general anesthetics (GAs and anti-epileptics (AEDs, mimic alcohol in triggering widespread apoptotic death of neurons and OLs in the third trimester-equivalent animal brain, and that human children exposed to GAs during early infancy, or to AEDs during the third trimester of gestation, have a significantly increased incidence of FASD-like neurobehavioral disturbances. These findings provide evidence that exposure of the developing human brain to GAs in early infancy, or to alcohol or AEDs in late gestation, can cause FASD-like neurodevelopmental disability syndromes. We propose that the mechanism by which

  14. Disruptive innovations

    OpenAIRE

    Viglia, Giampaolo; H. Werthner; Buhalis, Dimitrios

    2016-01-01

    The diffusion of disrupting innovations has generated significant market changes, modifying the dominant logic and affecting the strategic positioning of companies. This structural change is affecting market structure, the networks and the services that tourism players are supposed to use (Gretzel et al. 2015). One can also refer to the notion of digital infrastructure, which provides a nice framework that connects the different stakeholders, their relations as well as internal dynamics. At t...

  15. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

    Directory of Open Access Journals (Sweden)

    Jonathan Nzakizwanayo

    Full Text Available Escherichia coli Nissle 1917 (EcN is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants, prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB, or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC, were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

  16. Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization

    Directory of Open Access Journals (Sweden)

    McDonald Karen

    2011-08-01

    Full Text Available Abstract Background Direct gene synthesis is becoming more popular owing to decreases in gene synthesis pricing. Compared with using natural genes, gene synthesis provides a good opportunity to optimize gene sequence for specific applications. In order to facilitate gene optimization, we have developed a stand-alone software called Visual Gene Developer. Results The software not only provides general functions for gene analysis and optimization along with an interactive user-friendly interface, but also includes unique features such as programming capability, dedicated mRNA secondary structure prediction, artificial neural network modeling, network & multi-threaded computing, and user-accessible programming modules. The software allows a user to analyze and optimize a sequence using main menu functions or specialized module windows. Alternatively, gene optimization can be initiated by designing a gene construct and configuring an optimization strategy. A user can choose several predefined or user-defined algorithms to design a complicated strategy. The software provides expandable functionality as platform software supporting module development using popular script languages such as VBScript and JScript in the software programming environment. Conclusion Visual Gene Developer is useful for both researchers who want to quickly analyze and optimize genes, and those who are interested in developing and testing new algorithms in bioinformatics. The software is available for free download at http://www.visualgenedeveloper.net.

  17. Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization.

    Science.gov (United States)

    Jung, Sang-Kyu; McDonald, Karen

    2011-08-16

    Direct gene synthesis is becoming more popular owing to decreases in gene synthesis pricing. Compared with using natural genes, gene synthesis provides a good opportunity to optimize gene sequence for specific applications. In order to facilitate gene optimization, we have developed a stand-alone software called Visual Gene Developer. The software not only provides general functions for gene analysis and optimization along with an interactive user-friendly interface, but also includes unique features such as programming capability, dedicated mRNA secondary structure prediction, artificial neural network modeling, network & multi-threaded computing, and user-accessible programming modules. The software allows a user to analyze and optimize a sequence using main menu functions or specialized module windows. Alternatively, gene optimization can be initiated by designing a gene construct and configuring an optimization strategy. A user can choose several predefined or user-defined algorithms to design a complicated strategy. The software provides expandable functionality as platform software supporting module development using popular script languages such as VBScript and JScript in the software programming environment. Visual Gene Developer is useful for both researchers who want to quickly analyze and optimize genes, and those who are interested in developing and testing new algorithms in bioinformatics. The software is available for free download at http://www.visualgenedeveloper.net.

  18. Development of Quality Management Systems: How Have Disruptive Technological Innovations in Quality Management Affected Organizations?

    Directory of Open Access Journals (Sweden)

    Pasi Hellman

    2013-07-01

    Full Text Available This research investigates the history of quality and development of quality management systems. History of quality is investigated from Hammurabi’s law all the way to today’s reigning quality initiatives including ISO, Six Sigma and Lean manufacturing. In the last chapters the current state of quality management is being examined. The foresight method is also opened up and examined. The Innovator’s dilemma concept, originally presented by Clayton Christensen, is used to analyse how quality management systems have been evolved in the last 100 years. Special emphasis is placed on the US manufacturing in the 1970s and 1980s. Based on the literature, the concept of Innovator’s dilemma and the fall of US manufacturing are merged. The result is a prime example that the Innovator’s Dilemma is a universal phenomenon. The industry leader is trying to maintain its position and do everything right but still it is destined to fail. The causes and effects are being discussed in later chapters.

  19. Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

    Science.gov (United States)

    Killian, Tobias; Dickopf, Steffen; Haas, Alexander K; Kirstenpfad, Claudia; Mayer, Klaus; Brinkmann, Ulrich

    2017-11-13

    We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.

  20. Endocrine disrupting chemicals

    DEFF Research Database (Denmark)

    Mandrup, Karen

    suggested as particularly sensitive to endocrine disruption. Mammary gland examination in toxicological studies may be useful for improving knowledge on possible influences of EDCs on human mammary glands and also be useful for detection of endocrine disrupting effects of chemicals as part of safety testing....... To improve knowledge on possible influences of endocrine disrupters on female reproductive system, the effects of EDCs on genital malformations in females and the development of mammary glands were studied in the present project. AIMS: The aims for the studies on male and female mammary gland development...... effects on prepubertal female rat mammary glands were observed at lower levels than those affecting other endpoints studied. CONCLUSION: The present findings in rats suggest that EDCs may affect mammary gland development in women and men, although risk assessment including comparison with exposure...

  1. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin

  2. Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development

    Energy Technology Data Exchange (ETDEWEB)

    Nayeb-Hashemi, Hamed; Desai, Anal; Demchev, Valeriy [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Bronson, Roderick T. [Department of Microbiology and Immunology, Harvard Medical School, Boston, MA 02115 (United States); Hornick, Jason L. [Department of Pathology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Cohen, David E. [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Ukomadu, Chinweike, E-mail: cukomadu@partners.org [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2015-09-18

    Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC. - Highlights: • Fgl1 knockout mice (Fgl1KO) are more prone to carcinogen-induced liver cancer compared to wild type (WT) mates. • Tumors from the Fgl1KO are molecularly distinct with enhanced Akt and mTOR activity in comparison with Fgl1WT tumors. • Tumors from the Fgl1KO have enhanced expression of Trim35 and Tnfrsf10b, putative HCC tumor suppressors.

  3. Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

    Science.gov (United States)

    Miyazawa, Ken; Yoshimi, Akira; Zhang, Silai; Sano, Motoaki; Nakayama, Mayumi; Gomi, Katsuya; Abe, Keietsu

    2016-09-01

    Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.

  4. Correlation of Endocrine Disrupting Chemicals Serum Levels and White Blood Cells Gene Expression of Nuclear Receptors in a Population of Infertile Women

    Directory of Open Access Journals (Sweden)

    Donatella Caserta

    2013-01-01

    Full Text Available Significant evidence supports that many endocrine disrupting chemicals could affect female reproductive health. Aim of this study was to compare the internal exposure to bisphenol A (BPA, perfluorooctane sulphonate (PFOS, perfluorooctanoic acid (PFOA, monoethylhexyl phthalate (MEHP, and di(2-ethylhexyl phthalate (DEHP in serum samples of 111 infertile women and 44 fertile women. Levels of gene expression of nuclear receptors (ERα, ERβ, AR, AhR, PXR, and PPARγ were also analyzed as biomarkers of effective dose. The percentage of women with BPA concentrations above the limit of detection was significantly higher in infertile women than in controls. No statistically significant difference was found with regard to PFOS, PFOA, MEHP and DEHP. Infertile patients showed gene expression levels of ERα, ERβ, AR, and PXR significantly higher than controls. In infertile women, a positive association was found between BPA and MEHP levels and ERα, ERβ, AR, AhR, and PXR expression. PFOS concentration positively correlated with AR and PXR expression. PFOA levels negatively correlated with AhR expression. No correlation was found between DEHP levels and all evaluated nuclear receptors. This study underlines the need to provide special attention to substances that are still widely present in the environment and to integrate exposure measurements with relevant indicators of biological effects.

  5. Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

    Science.gov (United States)

    Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

    2017-01-01

    Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51-/-) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51-/- and Rorc-/- expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51-/- females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

  6. Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein.

    Science.gov (United States)

    Zhang, Lian-Dong; Deng, Qian; Wang, Zi-Ming; Gao, Ming; Wang, Lei; Chong, Tie; Li, He-Cheng

    2013-01-01

    Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on

  7. Circadian clock genes in Drosophila: recent developments.

    Science.gov (United States)

    Subramanian, P; Balamurugan, E; Suthakar, G

    2003-08-01

    Circadian rhythms provide a temporal framework to living organisms and are established in a majority of eukaryotes and in a few prokaryotes. The molecular mechanisms of circadian clock is constantly being investigated in Drosophila melanogaster. The core of the clock mechanism was described by a transcription-translation feedback loop model involving period (per), timeless (tim), dclock and cycle genes. However, recent research has identified multiple feedback loops controlling rhythm generation and expression. Novel mutations of timeless throw more light on the functions of per and tim products. Analysis of pdf neuropeptide gene (expressed in circadian pacemaker cells in Drosophila), indicate that PDF acts as the principal circadian transmitter and is involved in output pathways. The product of cryptochrome is known to function as a circadian photoreceptor as well as component of the circadian clock. This review focuses on the recent progress in the field of molecular rhythm research in the fruit fly. The gene(s) and the gene product(s) that are involved in the transmission of environmental information to the clock, as well as the timing signals from the clock outward to cellular functions are remain to be determined.

  8. The prevalence of at-risk development in children 30 to 60 months old presenting with disruptive behaviors.

    Science.gov (United States)

    Szczepaniak, Dorota; McHenry, Megan S; Nutakki, Kavitha; Bauer, Nerissa S; Downs, Stephen M

    2013-10-01

    This study assessed the prevalence of failed developmental screens in 30- to 60-month-old children who presented with a behavioral complaint of disruptive behavior to a referral clinic. A retrospective chart review was performed in a specialty behavioral pediatric clinic. Outcome measures were obtained from children referred to the clinic with parental concern of disruptive behavior. Out of 151 patients, 66% (99/151) had been screened for developmental delays with a formal screening instrument. Of these, 71% (70/99) failed developmental screening in one or more domains. Preschoolers referred to a behavioral clinic for parental concerns of disruptive behaviors were more than 4 times more likely to be at risk for developmental delays than the general population, with a majority of suspected delays in communication and problem-solving domains. This finding suggests that developmental screening should be a part of the initial workup for children with disruptive behaviors.

  9. Sequential gene activation and gene imprinting during early embryo development in maize.

    Science.gov (United States)

    Meng, Dexuan; Zhao, Jianyu; Zhao, Cheng; Luo, Haishan; Xie, Mujiao; Liu, Renyi; Lai, Jinsheng; Zhang, Xiaolan; Jin, Weiwei

    2017-11-24

    Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in maize embryo remains controversial. Here, we used high throughput RNA sequencing on laser capture microdissection (LCM) and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3 to 13 days after pollination) to analyze allelic gene expression patterns. Co-expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos and a greater number of imprinted genes were identified at early embryo stages. Sixty-four strongly imprinted genes were identified (at the threshold of 9:1) on 5 DAP to 13 DAP manually dissected embryos (more imprinted genes on 5 DAP). Forty-one strongly imprinted genes were identified from 3 DAP and 5 DAP embryos obtained by LCM (more imprinted genes on 3DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperms and nor in embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryotic tissue. Our results shed lights on early maize embryo development and provide evidence supporting that gene imprinting occurs in maize embryos. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Disruption of genital ridge development causes aberrant primordial germ cell proliferation but does not affect their directional migration.

    Science.gov (United States)

    Chen, Su-Ren; Zheng, Qiao-Song; Zhang, Yang; Gao, Fei; Liu, Yi-Xun

    2013-03-05

    The directional migration and the following development of primordial germ cells (PGCs) during gonad formation are key steps for germline development. It has been proposed that the interaction between germ cells and genital ridge (GR) somatic cells plays essential roles in this process. However, the in vivo functional requirements of GR somatic cells in germ cell development are largely unknown. Wt1 mutation (Wt1(R394W/R394W)) results in GR agenesis through mitotic arrest of coelomic epitheliums. In this study, we employed the GR-deficient mouse model, Wt1(R394W/R394W), to investigate the roles of GR somatic cells in PGC migration and proliferation. We found that the number of PGCs was dramatically reduced in GR-deficient embryos at embryonic day (E) 11.5 and E12.5 due to decreased proliferation of PGCs, involving low levels of BMP signaling. In contrast, the germ cells in Wt1(R394W/R394W) embryos were still mitotically active at E13.5, while all the germ cells in control embryos underwent mitotic arrest at this stage. Strikingly, the directional migration of PGCs was not affected by the absence of GR somatic cells. Most of the PGCs reached the mesenchyme under the coelomic epithelium at E10.5 and no ectopic PGCs were noted in GR-deficient embryos. However, the precise positioning of PGCs was disrupted. Our work provides in vivo evidence that the proliferation of germ cells is precisely regulated by GR somatic cells during different stages of gonad development. GR somatic cells are probably dispensable for the directional migration of PGCs, but they are required for precise positioning of PGCs at the final step of migration.

  11. Genes regulating hypothalamic and pituitary development.

    Science.gov (United States)

    Parks, J S; Adess, M E; Brown, M R

    1997-11-01

    Several pituitary transcription factors have been identified in the last 3 years. They offer new insights into the processes that direct organogenesis, cell commitment, proliferation and differentiated function. All are DNA-binding proteins, but they have ties to different families of homeodomain proteins. They differ in their distribution and in the timing of their appearance and extinction. The Rathke's pouch homeobox protein (Rpx) has a paired-like homeodomain. In mice, it appears on embryonic day 8.5 (day e8.5) and is gone by day e14.5. Its targets for activation are unknown. Pituitary OTX has a tryptophan--phenylalanine--lysine motif in its homeodomain. It appears early and persists. It shows independent activation of the alpha-glycoprotein subunit (alpha-GSU) and pro-opiomelanocortin genes and co-operates with Pit-1 in activation of the growth hormone and prolactin genes. Pituitary Lim (P-Lim) protein also acts independently on the alpha-GSU gene, and acts in concert with Pit-1 to activate other genes. A fourth protein, termed the 'Prophet of Pit-1', or Prop-1, is the recently discovered cause of Ames dwarfism in mice. This paired-like protein is necessary for the subsequent expression of Pit-1 in somatotrophs, lactotrophs and thyrotrophs. Any or all of the newly discovered pituitary genes are candidates for mutations causing hypopituitarism in humans. As several are expressed transiently in tissues other than the pituitary during organogenesis, the phenotypes produced by mutations in these genes may prove to be complex.

  12. Circadian clock genes, ovarian development and diapause

    Directory of Open Access Journals (Sweden)

    Bradshaw William E

    2010-09-01

    Full Text Available Abstract Insects, like most organisms, have an internal circadian clock that oscillates with a daily rhythmicity, and a timing mechanism that mediates seasonal events, including diapause. In research published in BMC Biology, Ikeno et al. show that downregulation of the circadian clock genes period and cycle affects expression of ovarian diapause in the insect Riptortus pedestris. They interpret these important results as support for Erwin Bünning's (1936 hypothesis that the circadian clock constitutes the basis of photoperiodism. However, their observations could also be the result of pleiotropic effects of the individual clock genes. See research article http://www.biomedcentral.com/1741-7007/8/116

  13. Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice.

    Directory of Open Access Journals (Sweden)

    Kamfai Chan

    Full Text Available Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future.

  14. Vitamin D related genes in lung development and asthma pathogenesis.

    Science.gov (United States)

    Kho, Alvin T; Sharma, Sunita; Qiu, Weiliang; Gaedigk, Roger; Klanderman, Barbara; Niu, Simin; Anderson, Chris; Leeder, James S; Weiss, Scott T; Tantisira, Kelan G

    2013-11-05

    Poor maternal vitamin D intake is a risk factor for subsequent childhood asthma, suggesting that in utero changes related to vitamin D responsive genes might play a crucial role in later disease susceptibility. We hypothesized that vitamin D pathway genes are developmentally active in the fetal lung and that these developmental genes would be associated with asthma susceptibility and regulation in asthma. Vitamin D pathway genes were derived from PubMed and Gene Ontology surveys. Principal component analysis was used to identify characteristic lung development genes. Vitamin D regulated genes were markedly over-represented in normal human (odds ratio OR 2.15, 95% confidence interval CI: 1.69-2.74) and mouse (OR 2.68, 95% CI: 2.12-3.39) developing lung transcriptomes. 38 vitamin D pathway genes were in both developing lung transcriptomes with >63% of genes more highly expressed in the later than earlier stages of development. In immortalized B-cells derived from 95 asthmatics and their unaffected siblings, 12 of the 38 (31.6%) vitamin D pathway lung development genes were significantly differentially expressed (OR 3.00, 95% CI: 1.43-6.21), whereas 11 (29%) genes were significantly differentially expressed in 43 control versus vitamin D treated immortalized B-cells from Childhood Asthma Management Program subjects (OR 2.62, 95% CI: 1.22-5.50). 4 genes, LAMP3, PIP5K1B, SCARB2 and TXNIP were identified in both groups; each displays significant biologic plausibility for a role in asthma. Our findings demonstrate a significant association between early lung development and asthma-related phenotypes for vitamin D pathway genes, supporting a genomic mechanistic basis for the epidemiologic observations relating maternal vitamin D intake and childhood asthma susceptibility.

  15. Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.

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    Kamran Honarnejad

    Full Text Available Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER. Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.

  16. Development of gene expression assays measuring immune ...

    African Journals Online (AJOL)

    Whole blood from five Mycobacterium bovis-sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in ...

  17. Gene bionetwork analysis of ovarian primordial follicle development.

    Directory of Open Access Journals (Sweden)

    Eric E Nilsson

    2010-07-01

    Full Text Available Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2 and a new factor was identified, connective tissue growth factor (CTGF. CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction.

  18. Nipbl and mediator cooperatively regulate gene expression to control limb development.

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    Akihiko Muto

    2014-09-01

    Full Text Available Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS, the most common "cohesinopathy". It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb, knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.

  19. Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar.

    Science.gov (United States)

    Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

    2015-04-01

    Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5' and 3' flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. The tumor suppressor gene TRC8/RNF139 is disrupted by a constitutional balanced translocation t(8;22(q24.13;q11.21 in a young girl with dysgerminoma

    Directory of Open Access Journals (Sweden)

    Fiorio Patrizia

    2009-07-01

    Full Text Available Abstract Background RNF139/TRC8 is a potential tumor suppressor gene with similarity to PTCH, a tumor suppressor implicated in basal cell carcinomas and glioblastomas. TRC8 has the potential to act in a novel regulatory relationship linking the cholesterol/lipid biosynthetic pathway with cellular growth control and has been identified in families with hereditary renal (RCC and thyroid cancers. Haploinsufficiency of TRC8 may facilitate development of clear cell-RCC in association with VHL mutations, and may increase risk for other tumor types. We report a paternally inherited balanced translocation t(8;22 in a proposita with dysgerminoma. Methods The translocation was characterized by FISH and the breakpoints cloned, sequenced, and compared. DNA isolated from normal and tumor cells was checked for abnormalities by array-CGH. Expression of genes TRC8 and TSN was tested both on dysgerminoma and in the proposita and her father. Results The breakpoints of the translocation are located within the LCR-B low copy repeat on chromosome 22q11.21, containing the palindromic AT-rich repeat (PATRR involved in recurrent and non-recurrent translocations, and in an AT-rich sequence inside intron 1 of the TRC8 tumor-suppressor gene at 8q24.13. TRC8 was strongly underexpressed in the dysgerminoma. Translin is underexpressed in the dysgerminoma compared to normal ovary. TRC8 is a target of Translin (TSN, a posttranscriptional regulator of genes transcribed by the transcription factor CREM-tau in postmeiotic male germ cells. Conclusion A role for TRC8 in dysgerminoma may relate to its interaction with Translin. We propose a model in which one copy of TRC8 is disrupted by a palindrome-mediated translocation followed by complete loss of expression through suppression, possibly mediated by miRNA.

  1. Minimally disruptive medicine is needed for patients with multimorbidity: time to develop computerised medical record systems to meet this requirement.

    Science.gov (United States)

    Schattner, Peter; Barker, Fiona; de Lusignan, Simon

    2015-02-19

    Minimally disruptive medicine (MDM) is proposed as a method for more appropriately managing people with multiple chronic disease. Much clinical management is currently single disease focussed, with people with multimorbidity being managed according to multiple single disease guidelines. Current initiatives to improve care include education about individual conditions and creating an environment where multiple guidelines might be simultaneously supported. The patient-centred medical home (PCMH) is an example of the latter. However, educational programmes and PCMH may increase the burden on patients. The cumulative workload for patients in managing the impact of multiple disease-specific guidelines is only relatively recently recognised. There is an intellectual vacuum as to how best to manage multimorbidity and how informatics might support implementing MDM. There is currently no alternative to multiple single-condition- specific guidelines and a lack of certainty, should the treatment burden need to be reduced, as to which guideline might be 'dropped'. The best information about multimorbidity is recorded in primary care computerised medical record (CMR) systems and in an increasing number of integrated care organisations. CMR systems have the potential to flag individuals who might be in greatest need. However, CMR systems may also provide insights into whether there are ameliorating factors that might make it easier for them to be resilient to the burden of care. Data from such CMR systems might be used to develop the evidence base about how to better manage multimorbidity. There is potential for these information systems to help reduce the management burden on patients and clinicians. However, substantial investment in research-driven CMR development is needed if we are to achieve this.

  2. VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor.

    Science.gov (United States)

    Casper-Lindley, Catharina; Yildiz, Fitnat H

    2004-03-01

    Vibrio cholerae switches between smooth and rugose colonial variants. The rugose variant produces more vibrio polysaccharides (VPS(El Tor)) and forms well-developed biofilms. Both phenotypes depend on expression of vps biosynthesis genes. We identified a positive transcriptional regulator of vps gene expression, VpsT, which is homologous to response regulators of two-component regulatory systems. Disruption of vpsT in the rugose variant yields smooth colonies, prevents formation of mature biofilms, and decreases vps gene expression. The interaction between VpsT and VpsR, a previously identified positive regulator of vps genes, was also investigated.

  3. Emerging and Disruptive Technologies.

    Science.gov (United States)

    Kricka, Larry J

    2016-08-01

    Several emerging or disruptive technologies can be identified that might, at some point in the future, displace established laboratory medicine technologies and practices. These include increased automation in the form of robots, 3-D printing, technology convergence (e.g., plug-in glucose meters for smart phones), new point-of-care technologies (e.g., contact lenses with sensors, digital and wireless enabled pregnancy tests) and testing locations (e.g., Retail Health Clinics, new at-home testing formats), new types of specimens (e.g., cell free DNA), big biology/data (e.g., million genome projects), and new regulations (e.g., for laboratory developed tests). In addition, there are many emerging technologies (e.g., planar arrays, mass spectrometry) that might find even broader application in the future and therefore also disrupt current practice. One interesting source of disruptive technology may prove to be the Qualcomm Tricorder XPrize, currently in its final stages.

  4. A missense mutation in the APC tumor suppressor gene disrupts an ASF/SF2 splicing enhancer motif and causes pathogenic skipping of exon 14.

    Science.gov (United States)

    Gonçalves, Vânia; Theisen, Patrícia; Antunes, Ofélia; Medeira, Ana; Ramos, José Silva; Jordan, Peter; Isidro, Glória

    2009-03-09

    A missense mutation at codon 640 in the APC gene was identified in a familial adenomatous polyposis (FAP) patient, however, its pathological consequence remained unclear. Here we found that this missense mutation interferes at the nucleotide level with an exonic splicing regulatory element and leads to aberrant splicing of the mutant APC transcript rather than exerting its effect through the observed amino acid change. Analysis of the patient RNA revealed complete skipping of exon 14 in transcripts from the mutant APC allele, leading to a frameshift and a premature stop codon. When cloned into a splicing reporter minigene and transfected into colorectal cell lines, the exon 14 point mutation c.1918C>G (pR640G) was found sufficient to cause the observed exon skipping. Bioinformatic analysis predicted the mutation to change SRp55, hnRNP A1 or ASF/SF2 splicing factor binding sites. Using RNA interference methodology these predictions were experimentally validated and revealed that only ASF/SF2 was required for exon 14 inclusion. These research data identify APC mutation c.1918C>G (pR640G) as pathogenic and indicate a mechanism involving disruption of an ASF/SF2 exonic splicing enhancer element. The results allow genetic diagnosis of a hereditary tumour predisposition but also illustrate the need to complement in silico prediction by splicing reporter assays.

  5. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

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    Shoji Suzuki

    2017-01-01

    Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

  6. Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

    Science.gov (United States)

    Kissenpfennig, Adrien; Aït-Yahia, Smina; Clair-Moninot, Valérie; Stössel, Hella; Badell, Edgar; Bordat, Yann; Pooley, Joanne L.; Lang, Thierry; Prina, Eric; Coste, Isabelle; Gresser, Olivia; Renno, Toufic; Winter, Nathalie; Milon, Geneviève; Shortman, Ken; Romani, Nikolaus; Lebecque, Serge; Malissen, Bernard; Saeland, Sem; Douillard, Patrice

    2005-01-01

    Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin−/− mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin−/− mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin−/− LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin−/− mice were not impaired in their capacity to process native OVA protein for I-Ab-restricted presentation to CD4+ T lymphocytes or for H-2Kb-restricted cross-presentation to CD8+ T lymphocytes. langerin−/− mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin−/− and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin−/− C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG. PMID:15601833

  7. Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces Streptococcus mutans Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms.

    Science.gov (United States)

    Dashper, Stuart G; Catmull, Deanne V; Liu, Sze-Wei; Myroforidis, Helen; Zalizniak, Ilya; Palamara, Joseph E A; Huq, N Laila; Reynolds, Eric C

    2016-01-01

    Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

  8. Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces Streptococcus mutans Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms.

    Directory of Open Access Journals (Sweden)

    Stuart G Dashper

    Full Text Available Glass ionomer cements (GIC are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

  9. Gene expression profile data for mouse facial development.

    Science.gov (United States)

    Leach, Sonia M; Feng, Weiguo; Williams, Trevor

    2017-08-01

    This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009) [1] and "Systems Biology of facial development: contributions of ectoderm and mesenchyme" (Hooper et al., 2017 In press) [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press) [1], [2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  10. Gene expression profile data for mouse facial development

    Directory of Open Access Journals (Sweden)

    Sonia M. Leach

    2017-08-01

    Full Text Available This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009 [1] and “Systems Biology of facial development: contributions of ectoderm and mesenchyme” (Hooper et al., 2017 In press [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press [1,2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  11. Reinforcing the Egg-Timer: Recruitment of Novel Lophotrochozoa Homeobox Genes to Early and Late Development in the Pacific Oyster

    Science.gov (United States)

    Paps, Jordi; Xu, Fei; Zhang, Guofan; Holland, Peter W.H.

    2015-01-01

    The metazoan superclade Lophotrochozoa includes mollusks, annelids, and several other animal phyla. It is reasonable to assume that this organismal diversity may be traced, in part, to changes in developmentally important genes, such as the homeobox genes. Although most comparative studies have focussed on ancient homeobox gene families conserved across bilaterians, there are also “novel” homeobox genes that have arisen more recently in evolution, presumably by duplication followed by radical divergence and functional change. We classify 136 homeobox genes in the genome sequence of the Pacific oyster, Crassostrea gigas. The genome shows an unusually low degree of homeobox gene clustering, with disruption of the NK, Hox, and ParaHox gene clusters. Among the oyster genes, 31 do not fall into ancient metazoan or bilaterian homeobox gene families; we deduce that they originated in the lophotrochozoan clade. We compared eight lophotrochozoan genomes to trace the pattern of homeobox gene evolution across this clade, allowing us to define 19 new lophotrochozoan-specific clades within the ANTP, PRD, TALE, ZF, SIX, and CUT classes. Using transcriptome data, we compared temporal expression of each homeobox gene in oyster development, and discovered that the lophotrochozoan-specific homeobox genes have peak expression either in early development (egg to gastrula) or in late development (after the trochophore larval stage), but rarely in between. This finding is consistent with the egg-timer, hourglass or phylotypic stage model of developmental evolution, in which there is a conserved central phase of development, but more evolutionarily labile early and late phases. PMID:25631164

  12. Disruption of M-T5, a novel myxoma virus gene member of poxvirus host range superfamily, results in dramatic attenuation of myxomatosis in infected European rabbits.

    Science.gov (United States)

    Mossman, K; Lee, S F; Barry, M; Boshkov, L; McFadden, G

    1996-07-01

    Myxoma virus is a pathogenic poxvirus that induces a lethal myxomatosis disease profile in European rabbits, which is characterized by fulminating lesions at the primary site of inoculation, rapid dissemination to secondary internal organs and peripheral external sites, and supervening gram-negative bacterial infection. Here we describe the role of a novel myxoma virus protein encoded by the M-T5 open reading frame during pathogenesis. The myxoma virus M-T5 protein possesses no significant sequence homology to nonviral proteins but is a member of a larger poxviral superfamily designated host range proteins. An M-T5- mutant virus was constructed by disruption of both copies of the M-T5 gene followed by insertion of the selectable marker p7.5Ecogpt. Although the M-T5- deletion mutant replicated with wild-type kinetics in rabbit fibroblasts, infection of a rabbit CD4+ T-cell line (RL5) with the myxoma virus M-T5- mutant virus resulted in the rapid and complete cessation of both host and viral protein synthesis, accompanied by the manifestation of all the classical features of programmed cell death. Infection of primary rabbit peripheral mononuclear cells with the myxoma virus M-T5-mutant virus resulted in the apoptotic death of nonadherent lymphocytes but not adherent monocytes. Within the European rabbit, disruption of the M-T5 open reading frame caused a dramatic attenuation of the rapidly lethal myxomatosis infection, and none of the infected rabbits displayed any of the characteristic features of myxomatosis. The two most significant histological observations in rabbits infected with the M-T5-mutant virus were (i) the lack of progression of the infection past the primary site of inoculation, coupled with the establishment of a rapid and effective inflammatory reaction, and (ii) the inability of the virus to initiate a cellular reaction within secondary immune organs. We conclude that M-T5 functions as a critical virulence factor by allowing productive infection of

  13. Nodulin gene expression during soybean (Glycine max) nodule development.

    NARCIS (Netherlands)

    Gloudemans, T.; Vries, de S.; Bussink, H.J.; Malik, N.S.A.; Franssen, H.; Louwerse, J.; Bisseling, T.

    1987-01-01

    In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be

  14. Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

    Directory of Open Access Journals (Sweden)

    Qiusheng Kong

    Full Text Available Gene expression analysis in watermelon (Citrullus lanatus fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC, β-actin (ClACT, and alpha tubulin 5 (ClTUA5 as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1, a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

  15. Disruptive Behavior Disorders in Adolescents with ASD: Comparisons to Youth with Intellectual Disability or Typical Cognitive Development

    Science.gov (United States)

    Baker, Bruce L.; Blacher, Jan

    2015-01-01

    Dual diagnosis of autism spectrum disorder (ASD) and behavior problems and/or mental disorders has become increasingly recognized and studied. Reported rates in samples of mixed-age youth with ASD are often above 70%, making this comorbidity more the rule than the exception. The present study compared rates of disruptive behavior disorder…

  16. Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.

    Science.gov (United States)

    Liu, Zhongzhen; Cheng, Tina Tsz Kwan; Shi, Zhaoying; Liu, Ziran; Lei, Yong; Wang, Chengdong; Shi, Weili; Chen, Xiongfeng; Qi, Xufeng; Cai, Dongqing; Feng, Bo; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2016-01-01

    The RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development. We utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission. Total 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring. Our study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic

  17. Investigation of the effects of estrogen on skeletal gene expression during zebrafish larval head development

    Directory of Open Access Journals (Sweden)

    Ehsan Pashay Ahi

    2016-03-01

    Full Text Available The development of craniofacial skeletal structures requires well-orchestrated tissue interactions controlled by distinct molecular signals. Disruptions in normal function of these molecular signals have been associated with a wide range of craniofacial malformations. A pathway mediated by estrogens is one of those molecular signals that plays role in formation of bone and cartilage including craniofacial skeletogenesis. Studies in zebrafish have shown that while higher concentrations of 17-β estradiol (E2 cause severe craniofacial defects, treatment with lower concentrations result in subtle changes in head morphology characterized with shorter snouts and flatter faces. The molecular basis for these morphological changes, particularly the subtle skeletal effects mediated by lower E2 concentrations, remains unexplored. In the present study we address these effects at a molecular level by quantitative expression analysis of sets of candidate genes in developing heads of zebrafish larvae treated with two different E2 concentrations. To this end, we first validated three suitable reference genes, ppia2, rpl8 and tbp, to permit sensitive quantitative real-time PCR analysis. Next, we profiled the expression of 28 skeletogenesis-associated genes that potentially respond to estrogen signals and play role in craniofacial development. We found E2 mediated differential expression of genes involved in extracellular matrix (ECM remodelling, mmp2/9/13, sparc and timp2a, as well as components of skeletogenic pathways, bmp2a, erf, ptch1/2, rankl, rarab and sfrp1a. Furthermore, we identified a co-expressed network of genes, including cpn1, dnajc3, esr1, lman1, rrbp1a, ssr1 and tram1 with a stronger inductive response to a lower dose of E2 during larval head development.

  18. Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection.

    Science.gov (United States)

    Xu, Jintao; Eastman, Alison J; Flaczyk, Adam; Neal, Lori M; Zhao, Guolei; Carolan, Jacob; Malachowski, Antoni N; Stolberg, Valerie R; Yosri, Mohammed; Chensue, Stephen W; Curtis, Jeffrey L; Osterholzer, John J; Olszewski, Michal A

    2016-07-12

    Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4(+) T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance. Increased susceptibility to invasive fungal infections in patients on anti-TNF-α therapies underlines the need for understanding the cellular effects of TNF-α signaling in promoting protective immunity to fungal pathogens. Here, we demonstrate that early TNF-α signaling is required

  19. Roles of lignin biosynthesis and regulatory genes in plant development

    Science.gov (United States)

    Yoon, Jinmi; Choi, Heebak

    2015-01-01

    Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

  20. Clock Genes and Altered Sleep–Wake Rhythms: Their Role in the Development of Psychiatric Disorders

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    Annaëlle Charrier

    2017-04-01

    Full Text Available In mammals, the circadian clocks network (central and peripheral oscillators controls circadian rhythms and orchestrates the expression of a range of downstream genes, allowing the organism to anticipate and adapt to environmental changes. Beyond their role in circadian rhythms, several studies have highlighted that circadian clock genes may have a more widespread physiological effect on cognition, mood, and reward-related behaviors. Furthermore, single nucleotide polymorphisms in core circadian clock genes have been associated with psychiatric disorders (such as autism spectrum disorder, schizophrenia, anxiety disorders, major depressive disorder, bipolar disorder, and attention deficit hyperactivity disorder. However, the underlying mechanisms of these associations remain to be ascertained and the cause–effect relationships are not clearly established. The objective of this article is to clarify the role of clock genes and altered sleep–wake rhythms in the development of psychiatric disorders (sleep problems are often observed at early onset of psychiatric disorders. First, the molecular mechanisms of circadian rhythms are described. Then, the relationships between disrupted circadian rhythms, including sleep–wake rhythms, and psychiatric disorders are discussed. Further research may open interesting perspectives with promising avenues for early detection and therapeutic intervention in psychiatric disorders.

  1. Expression of Sox family genes in early lamprey development.

    Science.gov (United States)

    Uy, Benjamin R; Simoes-Costa, Marcos; Sauka-Spengler, Tatjana; Bronner, Marianne E

    2012-01-01

    Members of the Sox (Sry-related high mobility group box) family of transcription factors play a variety of roles during development of both vertebrates and invertebrates. A marked expansion in gene number occurred during the emergence of vertebrates, apparently via gene duplication events that are thought to have facilitated new functions. By screening a macroarrayed library as well as the lamprey genome, we have isolated genes of the Sox B, D, E and F subfamilies in the basal jawless vertebrate, lamprey. The expression patterns of all identified Sox genes were examined from gastrulation through early organogenesis (embryonic day 4-14), with particular emphasis on the neural crest, a vertebrate innovation. Coupled with phylogenetic analysis of these Sox genes, the results provide insight into gene duplication and di-vergence in paralog deployment occurring during early vertebrate evolution.

  2. Circadian rhythm disruption in cancer biology.

    Science.gov (United States)

    Savvidis, Christos; Koutsilieris, Michael

    2012-12-06

    Circadian rhythms show universally a 24-h oscillation pattern in metabolic, physiological and behavioral functions of almost all species. This pattern is due to a fundamental adaptation to the rotation of Earth around its own axis. Molecular mechanisms of generation of circadian rhythms organize a biochemical network in suprachiasmatic nucleus and peripheral tissues, building cell autonomous clock pacemakers. Rhythmicity is observed in transcriptional expression of a wide range of clock-controlled genes that regulate a variety of normal cell functions, such as cell division and proliferation. Desynchrony of this rhythmicity seems to be implicated in several pathologic conditions, including tumorigenesis and progression of cancer. In 2007, the International Agency for Research on Cancer (IARC) categorized "shiftwork that involves circadian disruption [as] probably carcinogenic to humans" (Group 2A in the IARC classification system of carcinogenic potency of an agentagent) (Painting, Firefighting, and Shiftwork; IARC; 2007). This review discusses the potential relation between disruptions of normal circadian rhythms with genetic driving machinery of cancer. Elucidation of the role of clockwork disruption, such as exposure to light at night and sleep disruption, in cancer biology could be important in developing new targeted anticancer therapies, optimizing individualized chronotherapy and modifying lighting environment in workplaces or homes.

  3. The α-Tubulin gene TUBA1A in Brain Development: A Key Ingredient in the Neuronal Isotype Blend

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    Jayne Aiken

    2017-09-01

    Full Text Available Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene TUBA1A, which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in TUBA1A.

  4. The α-Tubulin gene TUBA1A in Brain Development: A Key Ingredient in the Neuronal Isotype Blend.

    Science.gov (United States)

    Aiken, Jayne; Buscaglia, Georgia; Bates, Emily A; Moore, Jeffrey K

    2017-09-01

    Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene TUBA1A, which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in TUBA1A.

  5. Development of Gene Centric Modeling for Nutrient Cycling

    Science.gov (United States)

    opportunity to participate in the development of a gene-centric model to help predict potential changes in the biogeochemistry of aquatic ecosystems that may arise from anthropogenic stressors and management decisions

  6. T-Box Genes in Human Development and Disease.

    Science.gov (United States)

    Ghosh, T K; Brook, J D; Wilsdon, A

    2017-01-01

    T-box genes are important development regulators in vertebrates with specific patterns of expression and precise roles during embryogenesis. They encode transcription factors that regulate gene transcription, often in the early stages of development. The hallmark of this family of proteins is the presence of a conserved DNA binding motif, the "T-domain." Mutations in T-box genes can cause developmental disorders in humans, mostly due to functional deficiency of the relevant proteins. Recent studies have also highlighted the role of some T-box genes in cancer and in cardiomyopathy, extending their role in human disease. In this review, we focus on ten T-box genes with a special emphasis on their roles in human disease. © 2017 Elsevier Inc. All rights reserved.

  7. A novel mouse model of anterior segment dysgenesis (ASD: conditional deletion of Tsc1 disrupts ciliary body and iris development

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    Anna-Carin Hägglund

    2017-03-01

    Full Text Available Development of the cornea, lens, ciliary body and iris within the anterior segment of the eye involves coordinated interaction between cells originating from the ciliary margin of the optic cup, the overlying periocular mesenchyme and the lens epithelium. Anterior segment dysgenesis (ASD encompasses a spectrum of developmental syndromes that affect these anterior segment tissues. ASD conditions arise as a result of dominantly inherited genetic mutations and result in both ocular-specific and systemic forms of dysgenesis that are best exemplified by aniridia and Axenfeld–Rieger syndrome, respectively. Extensive clinical overlap in disease presentation amongst ASD syndromes creates challenges for correct diagnosis and classification. The use of animal models has therefore proved to be a robust approach for unravelling this complex genotypic and phenotypic heterogeneity. However, despite these successes, it is clear that additional genes that underlie several ASD syndromes remain unidentified. Here, we report the characterisation of a novel mouse model of ASD. Conditional deletion of Tsc1 during eye development leads to a premature upregulation of mTORC1 activity within the ciliary margin, periocular mesenchyme and lens epithelium. This aberrant mTORC1 signalling within the ciliary margin in particular leads to a reduction in the number of cells that express Pax6, Bmp4 and Msx1. Sustained mTORC1 signalling also induces a decrease in ciliary margin progenitor cell proliferation and a consequent failure of ciliary body and iris development in postnatal animals. Our study therefore identifies Tsc1 as a novel candidate ASD gene. Furthermore, the Tsc1-ablated mouse model also provides a valuable resource for future studies concerning the molecular mechanisms underlying ASD and acts as a platform for evaluating therapeutic approaches for the treatment of visual disorders.

  8. A novel mouse model of anterior segment dysgenesis (ASD): conditional deletion of Tsc1 disrupts ciliary body and iris development.

    Science.gov (United States)

    Hägglund, Anna-Carin; Jones, Iwan; Carlsson, Leif

    2017-03-01

    Development of the cornea, lens, ciliary body and iris within the anterior segment of the eye involves coordinated interaction between cells originating from the ciliary margin of the optic cup, the overlying periocular mesenchyme and the lens epithelium. Anterior segment dysgenesis (ASD) encompasses a spectrum of developmental syndromes that affect these anterior segment tissues. ASD conditions arise as a result of dominantly inherited genetic mutations and result in both ocular-specific and systemic forms of dysgenesis that are best exemplified by aniridia and Axenfeld-Rieger syndrome, respectively. Extensive clinical overlap in disease presentation amongst ASD syndromes creates challenges for correct diagnosis and classification. The use of animal models has therefore proved to be a robust approach for unravelling this complex genotypic and phenotypic heterogeneity. However, despite these successes, it is clear that additional genes that underlie several ASD syndromes remain unidentified. Here, we report the characterisation of a novel mouse model of ASD. Conditional deletion of Tsc1 during eye development leads to a premature upregulation of mTORC1 activity within the ciliary margin, periocular mesenchyme and lens epithelium. This aberrant mTORC1 signalling within the ciliary margin in particular leads to a reduction in the number of cells that express Pax6, Bmp4 and Msx1 Sustained mTORC1 signalling also induces a decrease in ciliary margin progenitor cell proliferation and a consequent failure of ciliary body and iris development in postnatal animals. Our study therefore identifies Tsc1 as a novel candidate ASD gene. Furthermore, the Tsc1-ablated mouse model also provides a valuable resource for future studies concerning the molecular mechanisms underlying ASD and acts as a platform for evaluating therapeutic approaches for the treatment of visual disorders. © 2017. Published by The Company of Biologists Ltd.

  9. Monocrotophos, an organophosphorus insecticide, disrupts the expression of HpNetrin and its receptor neogenin during early development in the sea urchin (Hemicentrotus pulcherrimus).

    Science.gov (United States)

    Zhang, Xiaona; Xu, Lei; Tian, Hua; Wang, Cuicui; Wang, Wei; Ru, Shaoguo

    2017-09-01

    Netrins, chemotropic guidance cues, can guide the extension of serotonergic axons by binding to netrin receptors during neural development. However, little is known about whether disruption of netrin signaling is involved in the mechanisms by which organophosphorus pesticides affect serotonergic nervous system (SNS) development. In this study, we evaluated the effects of the pesticide monocrotophos (MCP) on the expression patterns of HpNetrin and its receptor neogenin as well as on the intracellular calcium ion (Ca 2+ ) levels in Hemicentrotus pulcherrimus (sea urchin) by exposing fertilized embryos to 0, 0.01, 0.10, and 1.00mg/L MCP. The results showed that MCP disrupted HpNetrin and neogenin expression at different developmental stages in H. pulcherrimus and that Ca 2+ appeared to be involved in the MCP-induced developmental neurotoxicity. Specifically, the lower concentrations of MCP elevated HpNetrin and neogenin transcription, resulting in higher intracellular Ca 2+ levels during the early developmental stages in the sea urchin; this may affect netrin-directed cell migration/axon extension and subsequently disrupt serotonergic axon branching and synapse formation. In contrast, 1.00mg/L MCP exhibited an inhibitory effect on HpNetrin and neogenin transcription. This finding implies that the regulatory roles of these factors may be diminished during early development, thereby causing developmental defects in the sea urchin. Collectively, our results provide a basis for exploring the involvement of netrin and neogenin in the organophosphate-induced disruption of the SNS during development. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

    Science.gov (United States)

    Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru; Yamada, Osamu

    2017-03-01

    Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Third trimester-equivalent ethanol exposure is characterized by an acute cellular stress response and an ontogenetic disruption of genes critical for synaptic establishment and function in mice.

    Science.gov (United States)

    Kleiber, Morgan L; Laufer, Benjamin I; Stringer, Randa L; Singh, Shiva M

    2014-01-01

    The developing brain is remarkably sensitive to alcohol exposure, resulting in the wide range of cognitive and neurobehavioral characteristics categorized under the term fetal alcohol spectrum disorders (FASD). The brain is particularly susceptible to alcohol during synaptogenesis, a process that occurs heavily during the third trimester and is characterized by the establishment and pruning of neural circuitry; however, the molecular response of the brain to ethanol during synaptogenesis has not been documented. To model a binge-like exposure during the third-trimester neurodevelopmental equivalent, neonate mice were given a high (5 g/kg over 2 h) dose of ethanol at postnatal day 7. Acute transcript changes within the brain were assessed using expression arrays and analyzed for associations with gene ontology functional categories, canonical pathways, and gene network interactions. The short-term effect of ethanol was characterized by an acute stress response and a downregulation of energetically costly cellular processes. Further, alterations to a number of genes with roles in synaptic transmission and hormonal signaling, particularly those associated with the neuroendocrine development and function, were evident. Ethanol exposure during synaptogenesis was also associated with altered histone deacetylase and microRNA transcript levels, suggesting that abnormal epigenetic patterning may maintain some of the persistent molecular consequences of developmental ethanol exposure. The results shed insight into the sensitivity of the brain to ethanol during the third-trimester equivalent and outline how ethanol-induced alterations to genes associated with neural connectivity may contribute to FASD phenotypes. © 2014 S. Karger AG, Basel.

  12. Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.

    Directory of Open Access Journals (Sweden)

    Bryna Erblich

    Full Text Available The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R. Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1(op gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.

  13. Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

    Science.gov (United States)

    Etgen, Anne M.; Dobrenis, Kostantin; Pollard, Jeffrey W.

    2011-01-01

    The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure. PMID:22046273

  14. Gene expression changes and early events in cotton fibre development.

    Science.gov (United States)

    Lee, Jinsuk J; Woodward, Andrew W; Chen, Z Jeffrey

    2007-12-01

    Cotton is the dominant source of natural textile fibre and a significant oil crop. Cotton fibres, produced by certain species in the genus Gossypium, are seed trichomes derived from individual cells of the epidermal layer of the seed coat. Cotton fibre development is delineated into four distinct and overlapping developmental stages: fibre initiation, elongation, secondary wall biosynthesis and maturation. Recent advances in gene expression studies are beginning to provide new insights into a better understanding of early events in cotton fibre development. Fibre cell development is a complex process involving many pathways, including various signal transduction and transcriptional regulation components. Several analyses using expressed sequence tags and microarray have identified transcripts that preferentially accumulate during fibre development. These studies, as well as complementation and overexpression experiments using cotton genes in arabidopsis and tobacco, indicate some similar molecular events between trichome development from the leaf epidermis and fibre development from the ovule epidermis. Specifically, MYB transcription factors regulate leaf trichome development in arabidopsis and may regulate seed trichome development in cotton. In addition, transcript profiling and ovule culture experiments both indicate that several phytohormones and other signalling pathways mediate cotton fibre development. Auxin and gibberellins promote early stages of fibre initiation; ethylene- and brassinosteroid-related genes are up-regulated during the fibre elongation phase; and genes associated with calmodulin and calmodulin-binding proteins are up-regulated in fibre initials. Additional genomic data, mutant and functional analyses, and genome mapping studies promise to reveal the critical factors mediating cotton fibre cell development.

  15. The Increasing Prevalence in Intersex Variation from Toxicological Dysregulation in Fetal Reproductive Tissue Differentiation and Development by Endocrine-Disrupting Chemicals

    Science.gov (United States)

    Rich, Alisa L.; Phipps, Laura M.; Tiwari, Sweta; Rudraraju, Hemanth; Dokpesi, Philip O.

    2016-01-01

    An increasing number of children are born with intersex variation (IV; ambiguous genitalia/hermaphrodite, pseudohermaphroditism, etc.). Evidence shows that endocrine-disrupting chemicals (EDCs) in the environment can cause reproductive variation through dysregulation of normal reproductive tissue differentiation, growth, and maturation if the fetus is exposed to EDCs during critical developmental times in utero. Animal studies support fish and reptile embryos exhibited IV and sex reversal when exposed to EDCs. Occupational studies verified higher prevalence of offspring with IV in chemically exposed workers (male and female). Chemicals associated with endocrine-disrupting ability in humans include organochlorine pesticides, poly-chlorinated biphenyls, bisphenol A, phthalates, dioxins, and furans. Intersex individuals may have concurrent physical disorders requiring lifelong medical intervention and experience gender dysphoria. An urgent need exists to determine which chemicals possess the greatest risk for IV and the mechanisms by which these chemicals are capable of interfering with normal physiological development in children. PMID:27660460

  16. The Increasing Prevalence in Intersex Variation from Toxicological Dysregulation in Fetal Reproductive Tissue Differentiation and Development by Endocrine-Disrupting Chemicals.

    Science.gov (United States)

    Rich, Alisa L; Phipps, Laura M; Tiwari, Sweta; Rudraraju, Hemanth; Dokpesi, Philip O

    2016-01-01

    An increasing number of children are born with intersex variation (IV; ambiguous genitalia/hermaphrodite, pseudohermaphroditism, etc.). Evidence shows that endocrine-disrupting chemicals (EDCs) in the environment can cause reproductive variation through dysregulation of normal reproductive tissue differentiation, growth, and maturation if the fetus is exposed to EDCs during critical developmental times in utero. Animal studies support fish and reptile embryos exhibited IV and sex reversal when exposed to EDCs. Occupational studies verified higher prevalence of offspring with IV in chemically exposed workers (male and female). Chemicals associated with endocrine-disrupting ability in humans include organochlorine pesticides, poly-chlorinated biphenyls, bisphenol A, phthalates, dioxins, and furans. Intersex individuals may have concurrent physical disorders requiring lifelong medical intervention and experience gender dysphoria. An urgent need exists to determine which chemicals possess the greatest risk for IV and the mechanisms by which these chemicals are capable of interfering with normal physiological development in children.

  17. [Characterization of Bacillus thuringiensis sigK disruption mutant and its influence on activation of cry3A promoter].

    Science.gov (United States)

    Du, Lixin; Wei, Juan; Han, Lili; Chen, Zhen; Zhang, Jie; Song, Fuping; Huang, Dafang

    2011-09-01

    To construct and characterize a sigK gene disruption mutant of Bacillus thuringiensis and to study influence of sigK gene disruption on the activation of cry3A gene promoter. We constructed the sigK gene disruption mutant HD delta sigK by inserting kanamycin resistance gene via homologous recombination. Scanning electron microscopy and spore formation analysis were used to detect the abilities of sporulation and crystal protein formation of both the mutant and the wild-type strain. SDS-PAGE analysis was used to detect the expression of crystal protein. Beta-galactosidase assay of cry3A'-lacZ gene fusion was performed to analyze the influence of sigK gene disruption on the activation of cry3A promoter. The growth curve showed that mutant grew slowly in late stationary phase compared to the wild-type strain. Scanning electron microscopy and spore formation analysis indicated that no spore was produced in sigK disruption mutant. SDS-PAGE results exhibited that the expression of cry gene was significantly decreased in the mutant. Beta-galactosidase assay showed that the activation of cry3A promoter was stronger in the mutant than that in HD-73 during late stationary phase, but the disruption of sigK gene had no significant influence on the production of Cry1Ac which was initiated by cry3A gene promoter. These results indicated that sigK gene was one of the essential genes during the sporulation of Bacillus thuringiensis, and influenced the expression of crystal protein. The expression of crystal protein which was initiated by cry3A gene promoter in sigK disruption mutant could be used to develop high-efficiency and safe biological pesticides.

  18. Understanding disruptive behavior problems in preschool children.

    Science.gov (United States)

    Breitenstein, Susan M; Hill, Carri; Gross, Deborah

    2009-02-01

    Disruptive behavior problems in young children are the number one reason for referral to mental health agencies. However, owing to difficulties differentiating clinically significant disruptive behaviors from typical development, a significant proportion of young children with disruptive behavior problems go unidentified and untreated. Research supports the existence of disruptive behavior disorders in young children, and early identification and treatment are critical to interrupt the trajectory of early problems to more significant and impairing difficulties. The purposes of this article were to identify and discuss disruptive behavior problems in preschool children and to introduce readers to current definitions of disruptive behavior problems and emotion regulation. A review of risk factors and underlying emotion and behavior regulation difficulties implicated in the development of disruptive behavior problems is provided. Furthermore, clinical implications for nurses in the identification of disruptive behavior problems in preschool children for pediatric nurses are discussed.

  19. Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

    Science.gov (United States)

    Sierro, Frederic; Biben, Christine; Martínez-Muñoz, Laura; Mellado, Mario; Ransohoff, Richard M.; Li, Meizhang; Woehl, Blanche; Leung, Helen; Groom, Joanna; Batten, Marcel; Harvey, Richard P.; Martínez-A, Carlos; Mackay, Charles R.; Mackay, Fabienne

    2007-01-01

    Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7−/− mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7−/− mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7−/− heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4. PMID:17804806

  20. Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

    Science.gov (United States)

    Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

    2013-01-01

    Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study

  1. The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo

    Directory of Open Access Journals (Sweden)

    Cinelli Paolo

    2008-11-01

    Full Text Available Abstract Background Glycosphingolipids (GSL are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The β1,3 N-acetylglucosaminyltransferase-V (B3gnt5 enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL. Results The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species. Conclusion The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound β3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.

  2. Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes

    Directory of Open Access Journals (Sweden)

    Pereira Gonçalo AG

    2009-08-01

    Full Text Available Abstract Background The hemibiotrophic fungus Moniliophthora perniciosa is the causal agent of Witches' broom, a disease of Theobroma cacao. The pathogen life cycle ends with the production of basidiocarps in dead tissues of the infected host. This structure generates millions of basidiospores that reinfect young tissues of the same or other plants. A deeper understanding of the mechanisms underlying the sexual phase of this fungus may help develop chemical, biological or genetic strategies to control the disease. Results Mycelium was morphologically analyzed prior to emergence of basidiomata by stereomicroscopy, light microscopy and scanning electron microscopy. The morphological changes in the mycelium before fructification show a pattern similar to other members of the order Agaricales. Changes and appearance of hyphae forming a surface layer by fusion were correlated with primordia emergence. The stages of hyphal nodules, aggregation, initial primordium and differentiated primordium were detected. The morphological analysis also allowed conclusions on morphogenetic aspects. To analyze the genes involved in basidiomata development, the expression of some selected EST genes from a non-normalized cDNA library, representative of the fruiting stage of M. perniciosa, was evaluated. A macroarray analysis was performed with 192 selected clones and hybridized with two distinct RNA pools extracted from mycelium in different phases of basidiomata formation. This analysis showed two groups of up and down-regulated genes in primordial phases of mycelia. Hydrophobin coding, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase genes were grouped among the up-regulated. In the down-regulated group relevant genes clustered coding calmodulin, lanosterol 14 alpha demethylase and PIM1. In addition, 12 genes with more detailed expression profiles were analyzed by RT-qPCR. One aegerolysin gene had a peak of expression in

  3. Gene network analysis: from heart development to cardiac therapy.

    Science.gov (United States)

    Ferrazzi, Fulvia; Bellazzi, Riccardo; Engel, Felix B

    2015-03-01

    Networks offer a flexible framework to represent and analyse the complex interactions between components of cellular systems. In particular gene networks inferred from expression data can support the identification of novel hypotheses on regulatory processes. In this review we focus on the use of gene network analysis in the study of heart development. Understanding heart development will promote the elucidation of the aetiology of congenital heart disease and thus possibly improve diagnostics. Moreover, it will help to establish cardiac therapies. For example, understanding cardiac differentiation during development will help to guide stem cell differentiation required for cardiac tissue engineering or to enhance endogenous repair mechanisms. We introduce different methodological frameworks to infer networks from expression data such as Boolean and Bayesian networks. Then we present currently available temporal expression data in heart development and discuss the use of network-based approaches in published studies. Collectively, our literature-based analysis indicates that gene network analysis constitutes a promising opportunity to infer therapy-relevant regulatory processes in heart development. However, the use of network-based approaches has so far been limited by the small amount of samples in available datasets. Thus, we propose to acquire high-resolution temporal expression data to improve the mathematical descriptions of regulatory processes obtained with gene network inference methodologies. Especially probabilistic methods that accommodate the intrinsic variability of biological systems have the potential to contribute to a deeper understanding of heart development.

  4. Dietary exposure to polybrominated diphenyl ether 47 (BDE-47) inhibits development and alters thyroid hormone-related gene expression in the brain of Xenopus laevis tadpoles.

    Science.gov (United States)

    Yost, Alexandra T; Thornton, Leah M; Venables, Barney J; Sellin Jeffries, Marlo K

    2016-12-01

    Few studies have investigated the thyroid-disrupting effects of polybrominated diphenyl ethers (PBDEs) across multiple levels of biological organization in anurans, despite their suitability for the screening of thyroid disruptors. Therefore, the present study evaluated the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on development, thyroid histology and thyroid hormone-related gene expression in Xenopus laevis exposed to 0 (control), 50 (low), 500 (medium) or 5000μg BDE-47/g food (high) for 21days. Only the high dose of BDE-47 hindered growth and development; however, thyroid hormone-associated gene expression was downregulated in the brains of tadpoles regardless of dose. These results show that BDE-47 disrupts thyroid hormone signaling at the molecular and whole-organism levels and suggest that gene expression in the brain is a more sensitive endpoint than metamorphosis. Furthermore, the altered gene expression patterns among BDE-47-exposed tadpoles provide insight into the mechanisms of PBDE-induced thyroid disruption and highlight the potential for PBDEs to act as neurodevelopmental toxicants. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Singing-driven gene expression in the developing songbird brain

    OpenAIRE

    Johnson, Frank; Whitney, Osceola

    2005-01-01

    Neural and behavioral development arises from an integration of genetic and environmental influences, yet specifying the nature of this interaction remains a primary problem in neuroscience. Here, we review molecular and behavioral studies that focus on the role of singing-driven gene expression during neural and vocal development in the male zebra finch (Taeniopygia guttata), a songbird that learns a species-typical vocal pattern during juvenile development by imitating an adult male tutor. ...

  6. The gene identification problem: An overview for developers

    Energy Technology Data Exchange (ETDEWEB)

    Fickett, J.W.

    1995-03-27

    The gene identification problem is the problem of interpreting nucleotide sequences by computer, in order to provide tentative annotation on the location, structure, and functional class of protein-coding genes. This problem is of self-evident importance, and is far from being fully solved, particularly for higher eukaryotes, Thus it is not surprising that the number of algorithm and software developers working in this area is rapidly increasing. The present paper is an overview of the field, with an emphasis on eukaryotes, for such developers.

  7. Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway.

    Science.gov (United States)

    Kang, Eunchai; Jiang, Danye; Ryu, Yun Kyoung; Lim, Sanghee; Kwak, Minhye; Gray, Christy D; Xu, Michael; Choi, Jun H; Junn, Sue; Kim, Jieun; Xu, Jing; Schaefer, Michele; Johns, Roger A; Song, Hongjun; Ming, Guo-Li; Mintz, C David

    2017-07-01

    Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

  8. Genes expressed during the development and ripening of watermelon fruit.

    Science.gov (United States)

    Levi, A; Davis, A; Hernandez, A; Wechter, P; Thimmapuram, J; Trebitsh, T; Tadmor, Y; Katzir, N; Portnoy, V; King, S

    2006-11-01

    A normalized cDNA library was constructed using watermelon flesh mRNA from three distinct developmental time-points and was subtracted by hybridization with leaf cDNA. Random cDNA clones of the watermelon flesh subtraction library were sequenced from the 5' end in order to identify potentially informative genes associated with fruit setting, development, and ripening. One-thousand and forty-six 5'-end sequences (expressed sequence tags; ESTs) were assembled into 832 non-redundant sequences, designated as "EST-unigenes". Of these 832 "EST-unigenes", 254 ( approximately 30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 "EST-unigenes" ( approximately 20%) correspond to genes with unknown function, whereas 410 "EST-unigenes" ( approximately 50%) correspond to genes with known function in other plant species. These "EST-unigenes" are mainly associated with metabolism, membrane transport, cytoskeleton synthesis and structure, cell wall formation and cell division, signal transduction, nucleic acid binding and transcription factors, defense and stress response, and secondary metabolism. This study provides the scientific community with novel genetic information for watermelon as well as an expanded pool of genes associated with fruit development in watermelon. These genes will be useful targets in future genetic and functional genomic studies of watermelon and its development.

  9. T-Box Genes in Drosophila Mesoderm Development.

    Science.gov (United States)

    Reim, I; Frasch, M; Schaub, C

    2017-01-01

    In Drosophila there are eight genes encoding transcription factors of the T-box family, which are known to exert a variety of crucial developmental functions during ectodermal patterning processes, neuronal cell specification, mesodermal tissue development, and the development of extraembryonic tissues. In this review, we focus on the prominent roles of Drosophila T-box genes in mesodermal tissues. First, we describe the contributions of brachyenteron (byn) and optomotor-blind-related-gene-1 (org-1) to the development of the visceral mesoderm. Second, we provide an overview on the functions of the three Dorsocross paralogs (Doc1-3) and the two Tbx20-related paralogs (midline and H15) during Drosophila heart development. Third, we portray the roles of org-1 and midline/H15 in the specification of individual body wall and organ-attached muscles, including the function of org-1 in the transdifferentiation of certain heart-attached muscles during metamorphosis. The functional analysis of these evolutionarily conserved T-box genes, along with their interactions with other types of transcription factors and various signaling pathways, has provided key insights into the regulation of Drosophila visceral mesoderm, muscle, and heart development. © 2017 Elsevier Inc. All rights reserved.

  10. Genomic approach to study floral development genes in Rosa sp.

    Science.gov (United States)

    Dubois, Annick; Remay, Arnaud; Raymond, Olivier; Balzergue, Sandrine; Chauvet, Aurélie; Maene, Marion; Pécrix, Yann; Yang, Shu-Hua; Jeauffre, Julien; Thouroude, Tatiana; Boltz, Véronique; Martin-Magniette, Marie-Laure; Janczarski, Stéphane; Legeai, Fabrice; Renou, Jean-Pierre; Vergne, Philippe; Le Bris, Manuel; Foucher, Fabrice; Bendahmane, Mohammed

    2011-01-01

    Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower.

  11. Current Concepts in Neuroendocrine Disruption

    Science.gov (United States)

    2014-01-01

    In the last few years, it has become clear that a wide variety of environmental contaminants have specific effects on neuroendocrine systems in fish, amphibians, birds and mammals. While it is beyond the scope of this review to provide a comprehensive examination of all of these neuroendocrine disruptors, we will focus on select representative examples. Organochlorine pesticides bioaccumulate in neuroendocrine areas of the brain that directly regulate GnRH neurons, thereby altering the expression of genes downstream of GnRH signaling. Organochlorine pesticides can also agonize or antagonize hormone receptors, adversely affecting crosstalk between neurotransmitter systems. The impacts of polychlorinated biphenyls are varied and in many cases subtle. This is particularly true for neuroedocrine and behavioral effects of exposure. These effects impact sexual differentiation of the hypothalamic-pituitary-gonadal axis, and other neuroendocrine systems regulating the thyroid, metabolic, and stress axes and their physiological responses. Weakly estrogenic and anti-androgenic pollutants such as bisphenol A, phthalates, phytochemicals, and the fungicide vinclozolin can lead to severe and widespread neuroendocrine disruptions in discrete brain regions, including the hippocampus, amygdala, and hypothalamus, resulting in behavioral changes in a wide range of species. Behavioral features that have been shown to be affected by one or more these chemicals include cognitive deficits, heightened anxiety or anxiety-like, sociosexual, locomotor, and appetitive behaviors. Neuroactive pharmaceuticals are now widely detected in aquatic environments and water supplies through the release of wastewater treatment plant effluents. The antidepressant fluoxetine is one such pharmaceutical neuroendocrine disruptor. Fluoxetine is a selective serotonin reuptake inhibitor that can affect multiple neuroendocrine pathways and behavioral circuits, including disruptive effects on reproduction and

  12. Adverse effects on sexual development in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides

    DEFF Research Database (Denmark)

    Hass, Ulla; Boberg, Julie; Christiansen, Sofie

    2012-01-01

    The present study investigated whether a mixture of low doses of five environmentally relevant endocrine disrupting pesticides, epoxiconazole, mancozeb, prochloraz, tebuconazole and procymidone, would cause adverse developmental toxicity effects in rats. In rat dams, a significant increase...... in gestation length was seen, while in male offspring increased nipple retention and increased incidence and severity of genital malformations were observed. Severe mixture effects on gestation length, nipple retention and genital malformations were seen at dose levels where the individual pesticides caused...... no or smaller effects when given alone. Generally, the mixture effect predictions based on dose-additivity were in good agreement with the observed effects. The results indicate that there is a need for modification of risk assessment procedures for pesticides, in order to take account of the mixture effects...

  13. Effects of tamoxifen on the sex determination gene and the activation of sex reversal in the developing gonad of mice.

    Science.gov (United States)

    Yu, Mingxi; Wang, Jingyun; Liu, Wei; Qin, Junwen; Zhou, Quan; Wang, Yongan; Huang, Huihui; Chen, Wenli; Ma, Chao

    2014-07-03

    Tamoxifen, as well as most endocrine-disrupting chemicals, affects the reproductive system and sexual development, but little is known about its disruption of the molecular pathways regulating mammalian sex determination. In fetal mice, the expression levels and pattern of key genes involved in controlling sexually dimorphic balance were analyzed both in vivo and in vitro by using whole-mount in situ hybridization and quantitative-PCR. Developmental tamoxifen exposure induced abnormal up-regulation of the testis differentiation marker Pdfgra in Leydig cells and of Sox9 and Fgf9 in Sertoli cells in XX gonad. Immunohistochemistry analysis confirmed the over-expression of SOX9 protein. Accordingly, the ovary development marker Foxl2 was depressed at both the mRNA and protein levels. The increase in testosterone and the reduction in 17β-estradiol and progesterone were observed by using the in vitro assay with organotypic cultures. Taken together, results indicated that tamoxifen induced the ectopic expression of well-established sex-specific genes during the critical developmental period, thus resulting in abnormal testicular development in the XX gonad of mammals. This study facilitates a better understanding of the molecular mechanisms of antiestrogens and possibly of compounds that interrupt estrogen signaling by other modes of action, and the association with the pathogenesis of human sexual developmental disorders. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings

    Science.gov (United States)

    Blancaflor, Elison B.; Hou, Guichuan; Chapman, Kent D.

    2003-01-01

    N-Acylethanolamines (NAEs) are prevalent in desiccated seeds of various plant species, and their levels decline substantially during seed imbibition and germination. Here, seeds of Arabidopsis thaliana (L.) Heynh. were germinated in, and seedlings maintained on, micromolar concentrations of N-lauroylethanolamine (NAE 12:0). NAE 12:0 inhibited root elongation, increased radial swelling of root tips, and reduced root hair numbers in a highly selective and concentration-dependent manner. These effects were reversible when seedlings were transferred to NAE-free medium. Older seedlings (14 days old) acclimated to exogenous NAE by increased formation of lateral roots, and generally, these lateral roots did not exhibit the severe symptoms observed in primary roots. Cells of NAE-treated primary roots were swollen and irregular in shape, and in many cases showed evidence, at the light- and electron-microscope levels, of improper cell wall formation. Microtubule arrangement was disrupted in severely distorted cells close to the root tip, and endoplasmic reticulum (ER)-localized green fluorescent protein (mGFP5-ER) was more abundant, aggregated and distributed differently in NAE-treated root cells, suggesting disruption of proper cell division, endomembrane organization and vesicle trafficking. These results suggest that NAE 12:0 likely influences normal cell expansion in roots by interfering with intracellular membrane trafficking to and/or from the cell surface. The rapid metabolism of NAEs during seed imbibition/germination may be a mechanism to remove this endogenous class of lipid mediators to allow for synchronized membrane reorganization associated with cell expansion.

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

    Directory of Open Access Journals (Sweden)

    Martinelli Federico

    2009-10-01

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

  16. The Arabidopsis thaliana homeobox gene ATHB12 is involved in symptom development caused by geminivirus infection.

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    Jungan Park

    Full Text Available BACKGROUND: Geminiviruses are single-stranded DNA viruses that infect a number of monocotyledonous and dicotyledonous plants. Arabidopsis is susceptible to infection with the Curtovirus, Beet severe curly top virus (BSCTV. Infection of Arabidopsis with BSCTV causes severe symptoms characterized by stunting, leaf curling, and the development of abnormal inflorescence and root structures. BSCTV-induced symptom development requires the virus-encoded C4 protein which is thought to interact with specific plant-host proteins and disrupt signaling pathways important for controlling cell division and development. Very little is known about the specific plant regulatory factors that participate in BSCTV-induced symptom development. This study was conducted to identify specific transcription factors that are induced by BSCTV infection. METHODOLOGY/PRINCIPAL FINDINGS: Arabidopsis plants were inoculated with BSCTV and the induction of specific transcription factors was monitored using quantitative real-time polymerase chain reaction assays. We found that the ATHB12 and ATHB7 genes, members of the homeodomain-leucine zipper family of transcription factors previously shown to be induced by abscisic acid and water stress, are induced in symptomatic tissues of Arabidopsis inoculated with BSCTV. ATHB12 expression is correlated with an array of morphological abnormalities including leaf curling, stunting, and callus-like structures in infected Arabidopsis. Inoculation of plants with a BSCTV mutant with a defective c4 gene failed to induce ATHB12. Transgenic plants expressing the BSCTV C4 gene exhibited increased ATHB12 expression whereas BSCTV-infected ATHB12 knock-down plants developed milder symptoms and had lower ATHB12 expression compared to the wild-type plants. Reporter gene studies demonstrated that the ATHB12 promoter was responsive to BSCTV infection and the highest expression levels were observed in symptomatic tissues where cell cycle genes also were

  17. Development of Viral Vectors for Gene Therapy for Chronic Pain

    Directory of Open Access Journals (Sweden)

    Yu Huang

    2011-01-01

    Full Text Available Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

  18. The role of genes, epigenetics and ontogeny in behavioural development

    NARCIS (Netherlands)

    Rodenburg, T.B.

    2014-01-01

    This review focuses on the role of genes, epigenetics and ontogeny in behavioural devel-opment of animals. The behavioural characteristics of an individual are determined by itsgenes and by its physical and social environment. Not only the individual’s early life andcurrent environment are of

  19. Gene expression during testis development in Duroc boars

    DEFF Research Database (Denmark)

    Lervik, Siri; Kristoffersen, Anja Bråthen; Conley, Lene

    2015-01-01

    Androstenone is a steroid pheromone occurring in the pubertal Leydig cells. Breeding against androstenone can decrease pheromone odour in swine meat but appears to cause unwanted side effects such as delayed onset of puberty. To study causality, global gene expression in developing boar testes...

  20. Gene expression profile of androgen modulated genes in the murine fetal developing lung

    Directory of Open Access Journals (Sweden)

    Côté Mélissa

    2010-01-01

    Full Text Available Abstract Background Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood. Methods To build up a better understanding of the effect of androgens on lung development, we analyzed by microarrays the expression of genes showing a sexual difference and those modulated by androgens. Lungs of murine fetuses resulting from a timely mating window of 1 hour were studied at gestational day 17 (GD17 and GD18, corresponding to the period of surge of surfactant production. Using injections of the antiandrogen flutamide to pregnant mice, we hunted for genes in fetal lungs which are transcriptionally modulated by androgens. Results Results revealed that 1844 genes were expressed with a sexual difference at GD17 and 833 at GD18. Many genes were significantly modulated by flutamide: 1597 at GD17 and 1775 at GD18. Datasets were analyzed by using in silico tools for reconstruction of cellular pathways. Between GD17 and GD18, male lungs showed an intensive transcriptional activity of proliferative pathways along with the onset of lung differentiation. Among the genes showing a sex difference or an antiandrogen modulation of their expression, we specifically identified androgen receptor interacting genes, surfactant related genes in particularly those involved in the pathway leading to phospholipid synthesis, and several genes of lung development regulator pathways. Among these latter, some genes related to Shh, FGF, TGF-beta, BMP, and Wnt signaling are modulated by sex and/or antiandrogen treatment. Conclusion Our results show clearly that there is a real delay in lung maturation between

  1. Role for the Wilms tumor gene in genital development

    Energy Technology Data Exchange (ETDEWEB)

    van Heyningen, V.; Bickmore, W.A.; Seawright, A.; Fletcher, J.M.; Maule, J.; Hastie, N.D. (Western General Hospital, Edinburgh (England)); Fekete, G. (Semmelweis Univ. Medical School, Budapest (Hungary)); Gessler, M.; Bruns, G.A.P. (Harvard Medical School, Boston, MA (USA)); Huerre-Jeanpierre, C.; Junien, C. (Institut National de la Sante et de la Recherche Medicale, Paris (France)); Williams, B.R.G. (Univ. of Toronto, Ontario (Canada))

    1990-07-01

    Detailed molecular definition of the WAGR region at chromosome 11p13 has been achieved by chromosome breakpoint analysis and long-range restriction mapping. Here the authors describe the molecular detection of a cytogenetically invisible 1-megabase deletion in an individual with aniridia, cryptorchidism, and hypospadias but no Wilms tumor (WT). The region of overlap between this deletion and one associated with WT and similar genital anomalies but no aniridia covers a region of 350-400 kilobases, which is coincident with the extent of homozygous deletion detected in tumor tissue from a sporadic WT. A candidate WT gene located within this region has recently been isolated, suggesting nonpenetrance for tumor expression in the first individual. The inclusion within the overlap region of a gene for WT predisposition and a gene for the best-documented WT-associated genitourinary malformations leads to suggest that both of these anomalies result from a loss-of-function mutation at the same locus. This in turn implies that the WT gene exerts pleiotropic effect on both kidney and genitourinary development, a possibility supported by the observed expression pattern of the WT candidate gene in developing kidney and gonads.

  2. From Digital Disruption to Business Model Scalability

    DEFF Research Database (Denmark)

    Nielsen, Christian; Lund, Morten; Thomsen, Peter Poulsen

    2017-01-01

    as a response to digital disruption. A series of case studies illustrate that besides frequent existing messages in the business literature relating to the importance of creating agile businesses, both in growing and declining economies, as well as hard to copy value propositions or value propositions that take......This article discusses the terms disruption, digital disruption, business models and business model scalability. It illustrates how managers should be using these terms for the benefit of their business by developing business models capable of achieving exponentially increasing returns to scale...... will seldom lead to business model scalability capable of competing with digital disruption(s)....

  3. Developing integrated crop knowledge networks to advance candidate gene discovery.

    Science.gov (United States)

    Hassani-Pak, Keywan; Castellote, Martin; Esch, Maria; Hindle, Matthew; Lysenko, Artem; Taubert, Jan; Rawlings, Christopher

    2016-12-01

    The chances of raising crop productivity to enhance global food security would be greatly improved if we had a complete understanding of all the biological mechanisms that underpinned traits such as crop yield, disease resistance or nutrient and water use efficiency. With more crop genomes emerging all the time, we are nearer having the basic information, at the gene-level, to begin assembling crop gene catalogues and using data from other plant species to understand how the genes function and how their interactions govern crop development and physiology. Unfortunately, the task of creating such a complete knowledge base of gene functions, interaction networks and trait biology is technically challenging because the relevant data are dispersed in myriad databases in a variety of data formats with variable quality and coverage. In this paper we present a general approach for building genome-scale knowledge networks that provide a unified representation of heterogeneous but interconnected datasets to enable effective knowledge mining and gene discovery. We describe the datasets and outline the methods, workflows and tools that we have developed for creating and visualising these networks for the major crop species, wheat and barley. We present the global characteristics of such knowledge networks and with an example linking a seed size phenotype to a barley WRKY transcription factor orthologous to TTG2 from Arabidopsis, we illustrate the value of integrated data in biological knowledge discovery. The software we have developed (www.ondex.org) and the knowledge resources (http://knetminer.rothamsted.ac.uk) we have created are all open-source and provide a first step towards systematic and evidence-based gene discovery in order to facilitate crop improvement.

  4. A provisional gene regulatory atlas for mouse heart development.

    Science.gov (United States)

    Chen, Hailin; VanBuren, Vincent

    2014-01-01

    Congenital Heart Disease (CHD) is one of the most common birth defects. Elucidating the molecular mechanisms underlying normal cardiac development is an important step towards early identification of abnormalities during the developmental program and towards the creation of early intervention strategies. We developed a novel computational strategy for leveraging high-content data sets, including a large selection of microarray data associated with mouse cardiac development, mouse genome sequence, ChIP-seq data of selected mouse transcription factors and Y2H data of mouse protein-protein interactions, to infer the active transcriptional regulatory network of mouse cardiac development. We identified phase-specific expression activity for 765 overlapping gene co-expression modules that were defined for obtained cardiac lineage microarray data. For each co-expression module, we identified the phase of cardiac development where gene expression for that module was higher than other phases. Co-expression modules were found to be consistent with biological pathway knowledge in Wikipathways, and met expectations for enrichment of pathways involved in heart lineage development. Over 359,000 transcription factor-target relationships were inferred by analyzing the promoter sequences within each gene module for overrepresentation against the JASPAR database of Transcription Factor Binding Site (TFBS) motifs. The provisional regulatory network will provide a framework of studying the genetic basis of CHD.

  5. A provisional gene regulatory atlas for mouse heart development.

    Directory of Open Access Journals (Sweden)

    Hailin Chen

    Full Text Available Congenital Heart Disease (CHD is one of the most common birth defects. Elucidating the molecular mechanisms underlying normal cardiac development is an important step towards early identification of abnormalities during the developmental program and towards the creation of early intervention strategies. We developed a novel computational strategy for leveraging high-content data sets, including a large selection of microarray data associated with mouse cardiac development, mouse genome sequence, ChIP-seq data of selected mouse transcription factors and Y2H data of mouse protein-protein interactions, to infer the active transcriptional regulatory network of mouse cardiac development. We identified phase-specific expression activity for 765 overlapping gene co-expression modules that were defined for obtained cardiac lineage microarray data. For each co-expression module, we identified the phase of cardiac development where gene expression for that module was higher than other phases. Co-expression modules were found to be consistent with biological pathway knowledge in Wikipathways, and met expectations for enrichment of pathways involved in heart lineage development. Over 359,000 transcription factor-target relationships were inferred by analyzing the promoter sequences within each gene module for overrepresentation against the JASPAR database of Transcription Factor Binding Site (TFBS motifs. The provisional regulatory network will provide a framework of studying the genetic basis of CHD.

  6. Gene Regulatory Network for Tapetum Development in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Dan-Dan Li

    2017-09-01

    Full Text Available In flowering plants, male gametophyte development occurs in the anther. Tapetum, the innermost of the four anther somatic layers, surrounds the developing reproductive cells to provide materials for pollen development. A genetic pathway of DYT1-TDF1-AMS-MS188 in regulating tapetum development has been proven. Here we used laser microdissection and pressure catapulting to capture and analyze the transcriptome data for the Arabidopsis tapetum at two stages. With a comprehensive analysis by the microarray data of dyt1, tdf1, ams, and ms188 mutants, we identified possible downstream genes for each transcription factor. These transcription factors regulate many biological processes in addition to activating the expression of the other transcription factor. Briefly, DYT1 may also regulate early tapetum development via E3 ubiquitin ligases and many other transcription factors. TDF1 is likely involved in redox and cell degradation. AMS probably regulates lipid transfer proteins, which are involved in pollen wall formation, and other E3 ubiquitin ligases, functioning in degradating proteins produced in previous processes. MS188 is responsible for most cell wall-related genes, functioning both in tapetum cell wall degradation and pollen wall formation. These results propose a more complex gene regulatory network for tapetum development and function.

  7. Genome engineering and gene expression control for bacterial strain development.

    Science.gov (United States)

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. CRISPR-Mediated Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ Triple Gene Disruption Reveals NKT Cell Defects but Not T Follicular Helper Cell Defects.

    Directory of Open Access Journals (Sweden)

    Joyce K Hu

    Full Text Available SAP (SH2D1A is required intrinsically in CD4 T cells to generate germinal center responses and long-term humoral immunity. SAP binds to SLAM family receptors, including SLAM, CD84, and Ly108 to enhance cytokine secretion and sustained T cell:B cell adhesion, which both improve T follicular helper (Tfh cell aid to germinal center (GC B cells. To understand the overlapping roles of multiple SLAM family receptors in germinal center responses, Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption (Slamf1,5,6Δ/Δ mice were generated using CRISPR-Cas9 gene editing to eliminate expression of SLAM (CD150, CD84, and Ly108, respectively. Gene targeting was highly efficient, with 6 of 6 alleles disrupted in 14 of 23 pups and the majority of alleles disrupted in the remaining pups. NKT cell differentiation in Slamf1,5,6Δ/Δ mice was defective, but not completely absent. The remaining NKT cells exhibited substantially increased 2B4 (SLAMF4 expression. Surprisingly, there were no overt defects in germinal center responses to acute viral infections or protein immunizations in Slamf1,5,6Δ/Δ mice, unlike Sh2d1a-/- mice. Similarly, in the context of a competitive environment, SLAM family receptor expressing GC Tfh cell, GC B cell, and plasma cell responses exhibited no advantages over Slamf1,5,6Δ/Δ cells.

  9. Singing-driven gene expression in the developing songbird brain.

    Science.gov (United States)

    Johnson, Frank; Whitney, Osceola

    2005-10-15

    Neural and behavioral development arises from an integration of genetic and environmental influences, yet specifying the nature of this interaction remains a primary problem in neuroscience. Here, we review molecular and behavioral studies that focus on the role of singing-driven gene expression during neural and vocal development in the male zebra finch (Taeniopygia guttata), a songbird that learns a species-typical vocal pattern during juvenile development by imitating an adult male tutor. A primary aim of our lab has been to identify naturally-occurring environmental influences that shape the propensity to sing. This ethological approach underlies our theoretical perspective, which is to integrate the significance of singing-driven gene expression into a broader ecological context.

  10. Circadian rhythms and gene expression during mouse molar tooth development.

    Science.gov (United States)

    Nirvani, Minou; Khuu, Cuong; Utheim, Tor Paaske; Hollingen, Henriette Stavik; Amundsen, Simon Furre; Sand, Lars Peter; Sehic, Amer

    2017-03-01

    Incremental markings in dental enamel suggest that the circadian clock may influence the molecular underpinnings orchestrating enamel formation. The aim of this study was to investigate whether the genes and microRNAs (miRNAs) oscillate in a circadian pattern during tooth and enamel development. Comparative gene and miRNA expression profiling of the first mandibular molar tooth germ isolated at different time-points during the light and night period was performed using microarrays and validated using real-time RT-PCR. Bioinformatic analysis was carried out using Ingenuity Pathway Analysis (IPA), and TargetScan software was used in order to identify computationally predicted miRNA-mRNA target relationships. In total, 439 genes and 32 miRNAs exhibited significantly different (p tooth germs. Genes involved in enamel formation, i.e. Amelx, Ambn, Amtn, and Odam, oscillated in a circadian pattern. Furthermore, the circadian clock genes, in particular Clock and Bmal1, oscillated in mouse molar tooth germ during 24-h intervals. The expression of Clock and Bmal1 was inversely correlated with the expression of miR-182 and miR-141, respectively. MiRNAs, including miR-182 and miR-141, are involved in the control of peripheral circadian rhythms in the developing tooth by regulating the expression of genes coding for circadian transcription factors such as CLOCK and BMAL1. Regulation of circadian rhythms may be important for enamel phenotype, and the morphology of dental enamel may vary between individuals due to differences in circadian profiles.

  11. Disorders of sex development: new genes, new concepts.

    Science.gov (United States)

    Ono, Makoto; Harley, Vincent R

    2013-02-01

    Formerly known as 'intersex' conditions, disorders of sex development (DSDs) are congenital conditions in which chromosomal, gonadal or anatomical sex is atypical. A complete revision of the nomenclature and classification of DSDs has been undertaken, which emphasizes the genetic aetiology of these disorders and discards pejorative terms. Uptake of the new terminology is widespread. DSDs affecting gonadal development are perhaps the least well understood. Unravelling the molecular mechanisms underlying gonadal development has revealed new causes of DSDs, although a specific molecular diagnosis is made in only ∼20% of patients. Conversely, identification of the molecular causes of DSDs has provided insight into the mechanisms of gonadal development. Studies of N-ethyl-N-nitrosourea mutagenesis in the mouse, and multigene diagnostic screening and genome-wide approaches, such as array-comparative genomic hybridization and next-generation sequencing, in patients with DSDs are accelerating the discovery of genes involved in gonadal development and DSDs. Furthermore, long-range gene regulatory mutations and multiple gene mutations are emerging as new causes of DSDs. Patients with DSDs, their parents and medical staff are confronted with challenging decisions regarding gender assignment, genital surgery and lifelong care. These advances are refining prognostic prediction and systematically improving the diagnosis and long-term management of children with DSDs.

  12. Development of a screening approach to detect thyroid disrupting chemicals that inhibit the human sodium iodide symporter (NIS).

    Science.gov (United States)

    Hallinger, Daniel R; Murr, Ashley S; Buckalew, Angela R; Simmons, Steven O; Stoker, Tammy E; Laws, Susan C

    2017-04-01

    The U.S. EPA's Endocrine Disruptor Screening Program aims to use high-throughput assays and computational toxicology models to screen and prioritize chemicals that may disrupt the thyroid signaling pathway. Thyroid hormone biosynthesis requires active iodide uptake mediated by the sodium/iodide symporter (NIS). Monovalent anions, such as the environmental contaminant perchlorate, are competitive inhibitors of NIS, yet limited information exists for more structurally diverse chemicals. A novel cell line expressing human NIS, hNIS-HEK293T-EPA, was used in a radioactive iodide uptake (RAIU) assay to identify inhibitors of NIS-mediated iodide uptake. The RAIU assay was optimized and performance evaluated with 12 reference chemicals comprising known NIS inhibitors and inactive compounds. An additional 39 chemicals including environmental contaminants were evaluated, with 28 inhibiting RAIU over 20% of that observed for solvent controls. Cell viability assays were performed to assess any confounding effects of cytotoxicity. RAIU and cytotoxic responses were used to calculate selectivity scores to group chemicals based on their potential to affect NIS. RAIU IC50 values were also determined for chemicals that displayed concentration-dependent inhibition of RAIU (≥50%) without cytotoxicity. Strong assay performance and highly reproducible results support the utilization of this approach to screen large chemical libraries for inhibitors of NIS-mediated iodide uptake. Published by Elsevier Ltd.

  13. Brd2 gene disruption causes ‘metabolically healthy’ obesity: Epigenetic and chromatin-based mechanisms that uncouple obesity from Type 2 diabetes

    Science.gov (United States)

    Wang, Fangnian; Deeney, Jude T.; Denis, Gerald V.

    2014-01-01

    Disturbed body energy balance can lead to obesity and obesity-driven diseases such as Type 2 diabetes, which have reached an epidemic level. Evidence indicates that obesity induced inflammation is a major cause of insulin resistance and Type 2 diabetes. Environmental factors, such as nutrients, affect body energy balance through epigenetic or chromatin-based mechanisms. As a bromodomain and external domain family transcription regulator, Brd2 regulates expression of many genes through interpretation of chromatin codes, and participates in the regulation of body energy balance and immune function. In the severely obese state, Brd2 knockdown in mice prevented obesity-induced inflammatory responses, protected animals from Type 2 diabetes, and thus uncoupled obesity from diabetes. Brd2 provides an important model for investigation of the function of transcription regulators and the development of obesity and diabetes; it also provides a possible target to treat obesity and diabetes through modulation of the function of a chromatin code reader. PMID:23374712

  14. The Role of Friends’ Disruptive Behavior in the Development of Children’s Tobacco Experimentation: Results from a Preventive Intervention Study

    Science.gov (United States)

    Huizink, Anja; Vuijk, Patricia

    2010-01-01

    Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends’ disruptive behavior as a mediator of the effects of a classroom based intervention on children’s tobacco experimentation. 433 Children (52% males) were randomly assigned to the Good Behavior Game (GBG) intervention, a universal preventive intervention targeting disruptive behavior, and facilitating positive prosocial peer interactions. Friends’ disruptive behavior was assessed from age 7–10 years. Participants’ experimentation with tobacco was assessed annually from age 10–13. Reduced rates in tobacco experimentation and friends’ disruptive behavior were found among GBG children, as compared to controls. Support for friends’ disruptive behavior as a mediator in the link between intervention status and tobacco experimentation was found. These results remained after controlling for friends’ and parental smoking status, and child ADHD symptoms. The results support the role of friends’ disruptive behavior in preadolescents’ tobacco experimentation. PMID:20694577

  15. Gene networks controlling the initiation of flower development.

    Science.gov (United States)

    Wellmer, Frank; Riechmann, José L

    2010-12-01

    The onset of flower formation is a key regulatory event during the life cycle of angiosperm plants, which marks the beginning of the reproductive phase of development. It has been shown that floral initiation is under tight genetic control, and deciphering the underlying molecular mechanisms has been a main area of interest in plant biology for the past two decades. Here, we provide an overview of the developmental and genetic processes that occur during floral initiation. We further review recent studies that have led to the genome-wide identification of target genes of key floral regulators and discuss how they have contributed to an in-depth understanding of the gene regulatory networks controlling early flower development. We focus especially on a master regulator of floral initiation in Arabidopsis thaliana APETALA1 (AP1), but also outline what is known about the AP1 network in other plant species and the evolutionary implications. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Endocrine Disrupting Chemicals (EDCs)

    Science.gov (United States)

    ... Pacientes y Cuidadores Hormones and Health The Endocrine System Hormones Endocrine Disrupting Chemicals (EDCs) Steroid and Hormone Abuse Peer ... and Health › Endocrine Disrupting Chemicals (EDCs) The Endocrine System Hormones Endocrine Disrupting Chemicals (EDCs) EDCs Myth vs. Fact Steroid ...

  17. Development of a fast liquid chromatography-tandem mass spectrometry method for the determination of endocrine-disrupting compounds in waters.

    Science.gov (United States)

    Di Carro, Marina; Scapolla, Carlo; Liscio, Camilla; Magi, Emanuele

    2010-09-01

    A fast liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed to study five endocrine-disrupting compounds (4-n-nonylphenol, bisphenol A, estrone, 17β-estradiol and 17α-ethinylestradiol) in water. Different columns were tested; the chromatographic separation of the analytes was optimized on a Pinnacle DB biphenylic column with a water-acetonitrile gradient elution, which allowed the separation of the selected endocrine-disrupting compounds (EDCs) in less than 6 min. Quantitative analysis was performed in selected reaction monitoring (SRM) mode; two transitions were chosen for each compound, using the most abundant for quantitation. Calibration curves using bisphenol A-d (16) as internal standard were drawn, showing good correlation coefficients (0.9993-0.9998). All figures of merit of the method were satisfactory; limits of detection were in the low pg range for all analytes. The method was then applied to the determination of the analytes in real water samples: to this aim, polar organic chemical integrative samplers (POCIS) were deployed in the influent and in the effluent of a drinking water treatment plant in Liguria (Italy). The EDC level was rather low in the influent and negligible in the outlet, reflecting the expected function of the treatment plant.

  18. Fiscal 2000 research and development of technologies for intelligent infrastructure creation and utilization. Development of high-precision screening assay system for endocrine disrupting chemicals and construction of database; 2000 nendo chiteki kiban sose riyo gijutsu kenkyu kaihatsu. Naibunpi kakuran busshitsu no koseido screening shiken hoho no kaihatsu oyobi data kiban seibi

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Research and development is carried out for the establishment of a system for assaying endocrine disruption now presenting itself as a hazard and for the application of the assay system to important chemicals and chemicals under development for the detection of presence of endocrine disrupting action in them. In this fiscal year, for the establishment of a reporter-gene assay system, cell characteristics were clarified through the determination of the expression sequence of the produced cell, the determination of the stable conservation period of the cell, and the study of the sustenance of activity. Studies were conducted, targeted at the stable supply of cells usable for high-throughput screening. Basic data were collected for the Hershberger assay being developed by OECD (Organization for Economic Cooperation and Development) and for the repeated administration test method for assaying thyroid hormone action by use of pubertal rats. For the assay of impact on environmental organisms, moreover, basic data were collected for the establishment of testing methods using fishes and amphibians. (NEDO)

  19. Conserved Gene Expression Programs in Developing Roots from Diverse Plants.

    Science.gov (United States)

    Huang, Ling; Schiefelbein, John

    2015-08-01

    The molecular basis for the origin and diversification of morphological adaptations is a central issue in evolutionary developmental biology. Here, we defined temporal transcript accumulation in developing roots from seven vascular plants, permitting a genome-wide comparative analysis of the molecular programs used by a single organ across diverse species. The resulting gene expression maps uncover significant similarity in the genes employed in roots and their developmental expression profiles. The detailed analysis of a subset of 133 genes known to be associated with root development in Arabidopsis thaliana indicates that most of these are used in all plant species. Strikingly, this was also true for root development in a lycophyte (Selaginella moellendorffii), which forms morphologically different roots and is thought to have evolved roots independently. Thus, despite vast differences in size and anatomy of roots from diverse plants, the basic molecular mechanisms employed during root formation appear to be conserved. This suggests that roots evolved in the two major vascular plant lineages either by parallel recruitment of largely the same developmental program or by elaboration of an existing root program in the common ancestor of vascular plants. © 2015 American Society of Plant Biologists. All rights reserved.

  20. FOXN1: a master regulator gene of thymic epithelial development programme

    Directory of Open Access Journals (Sweden)

    Rosa eRomano

    2013-07-01

    Full Text Available T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs drive T cell differentiation, education and selection processes, while the thymocyte-dependent signals allow TECs to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1 and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1 gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the postnatal thymus. An inborn null mutation in FOXN1 leads to Nude/SCID phenotype in mouse, rat and humans. In Foxn1-/- nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.

  1. Bisphenol a exposure disrupts genomic imprinting in the mouse.

    Directory of Open Access Journals (Sweden)

    Martha Susiarjo

    2013-04-01

    Full Text Available Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA. In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs including the Snrpn imprinting control region (ICR and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the

  2. Disruption of thyroid hormone (TH) levels and TH-regulated gene expression by polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and hydroxylated PCBs in e-waste recycling workers.

    Science.gov (United States)

    Zheng, Jing; He, Chun-Tao; Chen, She-Jun; Yan, Xiao; Guo, Mi-Na; Wang, Mei-Huan; Yu, Yun-Jiang; Yang, Zhong-Yi; Mai, Bi-Xian

    2017-05-01

    Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) are the primary toxicants released by electronic waste (e-waste) recycling, but their adverse effects on people working in e-waste recycling or living near e-waste sites have not been studied well. In the present study, the serum concentrations of PBDEs, PCBs, and hydroxylated PCBs, the circulating levels of thyroid hormones (THs), and the mRNA levels of seven TH-regulated genes in peripheral blood leukocytes of e-waste recycling workers were analyzed. The associations of the hormone levels and gene expression with the exposure to these contaminants were examined using multiple linear regression models. There were nearly no associations of the TH levels with PCBs and hydroxylated PCBs, whereas elevated hormone (T 4 and T 3 ) levels were associated with certain lower-brominated BDEs. While not statistically significant, we did observe a negative association between highly brominated PBDE congeners and thyroid-stimulating hormone (TSH) levels in the e-waste workers. The TH-regulated gene expression was more significantly associated with the organohalogen compounds (OHCs) than the TH levels in these workers. The TH-regulated gene expression was significantly associated with certain PCB and hydroxylated PCB congeners. However, the expression of most target genes was suppressed by PBDEs (mostly highly brominated congeners). This is the first evidence of alterations in TH-regulated gene expression in humans exposed to OHCs. Our findings indicated that OHCs may interfere with TH signaling and/or exert TH-like effects, leading to alterations in related gene expression in humans. Further research is needed to investigate the mechanisms of action and associated biological consequences of the gene expression disruption by OHCs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Transcription of the soybean leghemoglobin genes during nodule development

    DEFF Research Database (Denmark)

    Marcker, Anne; Lund, Marianne; Jensen, Erik Ø

    1984-01-01

    of the Lb(c1), Lb(c3) and Lb(a) genes while the transcription of the Lb(c2) gene is not amplified to a similar extent. All the Lb genes retain significant activity for a long period during the lifetime of a nodule. Consequently the soybean Lb genes are not regulated by a developmental gene switching...... mechanism as is the case for vertebrate globin genes. Concomitantly with the increase in Lb gene transcription some of the other nodule specific plant genes are activated. These specific changes in the activities of the Lb and nodulin genes precede the activation of the bacterial nitrogenase gene. Thus...

  4. Modeling the Drosophila gene cluster regulation network for muscle development.

    Science.gov (United States)

    Haye, Alexandre; Albert, Jaroslav; Rooman, Marianne

    2014-01-01

    The development of accurate and reliable dynamical modeling procedures that describe the time evolution of gene expression levels is a prerequisite to understanding and controlling the transcription process. We focused on data from DNA microarray time series for 20 Drosophila genes involved in muscle development during the embryonic stage. Genes with similar expression profiles were clustered on the basis of a translation-invariant and scale-invariant distance measure. The time evolution of these clusters was modeled using coupled differential equations. Three model structures involving a transcription term and a degradation term were tested. The parameters were identified in successive steps: network construction, parameter optimization, and parameter reduction. The solutions were evaluated on the basis of the data reproduction and the number of parameters, as well as on two biology-based requirements: the robustness with respect to parameter variations and the values of the expression levels not being unrealistically large upon extrapolation in time. Various solutions were obtained that satisfied all our evaluation criteria. The regulatory networks inferred from these solutions were compared with experimental data. The best solution has half of the experimental connections, which compares favorably with previous approaches. Biasing the network toward the experimental connections led to the identification of a model that is only slightly less good on the basis of the evaluation criteria. The non-uniqueness of the solutions and the variable agreement with experimental connections were discussed in the context of the different hypotheses underlying this type of approach.

  5. Identification of Osr2 Transcriptional Target Genes in Palate Development.

    Science.gov (United States)

    Fu, X; Xu, J; Chaturvedi, P; Liu, H; Jiang, R; Lan, Y

    2017-11-01

    Previous studies have identified the odd-skipped related 2 (Osr2) transcription factor as a key intrinsic regulator of palatal shelf growth and morphogenesis. However, little is known about the molecular program acting downstream of Osr2 in the regulation of palatogenesis. In this study, we isolated palatal mesenchyme cells from embryonic day 12.5 (E12.5) and E13.5 Osr2RFP/+ and Osr2RFP/- mutant mouse embryos and performed whole transcriptome RNA sequencing analyses. Differential expression analysis of the RNA sequencing datasets revealed that expression of 70 genes was upregulated and expression of 61 genes was downregulated by >1.5-fold at both E12.5 and E13.5 in the Osr2RFP/- palatal mesenchyme cells, in comparison with Osr2RFP/+ littermates. Gene ontology analysis revealed enrichment of signaling molecules and transcription factors crucial for skeletal development and osteoblast differentiation among those significantly upregulated in the Osr2 mutant palatal mesenchyme. Using quantitative real-time polymerase chain reaction (RT-PCR)and in situ hybridization assays, we validated that the Osr2-/- embryos exhibit significantly increased and expanded expression of many osteogenic pathway genes, including Bmp3, Bmp5, Bmp7, Mef2c, Sox6, and Sp7 in the developing palatal mesenchyme. Furthermore, we demonstrate that expression of Sema3a, Sema3d, and Sema3e, is ectopically activated in the developing palatal mesenchyme in Osr2-/- embryos. Through chromatin immunoprecipitation, followed by RT-PCR analysis, we demonstrate that endogenous Osr2 protein binds to the promoter regions of the Sema3a and Sema3d genes in the embryonic palatal mesenchyme. Moreover, Osr2 expression repressed the transcription from the Sema3a and Sema3d promoters in cotransfected cells. Since the Sema3 subfamily of signaling molecules plays diverse roles in the regulation of cell proliferation, migration, and differentiation, these data reveal a novel role for Osr2 in regulation of palatal

  6. Hox genes: choreographers in neural development, architects of circuit organization.

    Science.gov (United States)

    Philippidou, Polyxeni; Dasen, Jeremy S

    2013-10-02

    The neural circuits governing vital behaviors, such as respiration and locomotion, are comprised of discrete neuronal populations residing within the brainstem and spinal cord. Work over the past decade has provided a fairly comprehensive understanding of the developmental pathways that determine the identity of major neuronal classes within the neural tube. However, the steps through which neurons acquire the subtype diversities necessary for their incorporation into a particular circuit are still poorly defined. Studies on the specification of motor neurons indicate that the large family of Hox transcription factors has a key role in generating the subtypes required for selective muscle innervation. There is also emerging evidence that Hox genes function in multiple neuronal classes to shape synaptic specificity during development, suggesting a broader role in circuit assembly. This Review highlights the functions and mechanisms of Hox gene networks and their multifaceted roles during neuronal specification and connectivity. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Screening and analyzing genes associated with Amur tiger placental development.

    Science.gov (United States)

    Li, Q; Lu, T F; Liu, D; Hu, P F; Sun, B; Ma, J Z; Wang, W J; Wang, K F; Zhang, W X; Chen, J; Guan, W J; Ma, Y H; Zhang, M H

    2014-09-26

    The Amur tiger is a unique endangered species in the world, and thus, protection of its genetic resources is extremely important. In this study, an Amur tiger placenta cDNA library was constructed using the SMART cDNA Library Construction kit. A total of 508 colonies were sequenced, in which 205 (76%) genes were annotated and mapped to 74 KEGG pathways, including 29 metabolism, 29 genetic information processing, 4 environmental information processing, 7 cell motility, and 5 organismal system pathways. Additionally, PLAC8, PEG10 and IGF-II were identified after screening genes from the expressed sequence tags, and they were associated with placental development. These findings could lay the foundation for future functional genomic studies of the Amur tiger.

  8. Disruption of genital ridge development causes aberrant primordial germ cell proliferation but does not affect their directional migration

    OpenAIRE

    Chen, Su-Ren; Zheng, Qiao-Song; Zhang, Yang; Gao, Fei; Liu, Yi-Xun

    2013-01-01

    Background The directional migration and the following development of primordial germ cells (PGCs) during gonad formation are key steps for germline development. It has been proposed that the interaction between germ cells and genital ridge (GR) somatic cells plays essential roles in this process. However, the in vivo functional requirements of GR somatic cells in germ cell development are largely unknown. Results Wt1 mutation (Wt1 R394W/R394W) results in GR agenesis through mitotic arrest of...

  9. The Him gene inhibits the development of Drosophila flight muscles during metamorphosis.

    Science.gov (United States)

    Soler, Cédric; Taylor, Michael V

    2009-07-01

    During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.

  10. Brd2 gene disruption causes "metabolically healthy" obesity: epigenetic and chromatin-based mechanisms that uncouple obesity from type 2 diabetes.

    Science.gov (United States)

    Wang, Fangnian; Deeney, Jude T; Denis, Gerald V

    2013-01-01

    Disturbed body energy balance can lead to obesity and obesity-driven diseases such as Type 2 diabetes, which have reached an epidemic level. Evidence indicates that obesity-induced inflammation is a major cause of insulin resistance and Type 2 diabetes. Environmental factors, such as nutrients, affect body energy balance through epigenetic or chromatin-based mechanisms. As a bromodomain and external domain family transcription regulator, Brd2 regulates expression of many genes through interpretation of chromatin codes and participates in the regulation of body energy balance and immune function. In the severely obese state, Brd2 knockdown in mice prevented obesity-induced inflammatory responses, protected animals from insulin resistance, glucose intolerance and pancreatic beta cell dysfunction, and thus uncoupled obesity from diabetes. Brd2 provides an important model for investigation of the function of transcription regulators and the development of obesity and diabetes; it also provides a possible, innovative target to treat obesity and diabetes through modulation of the function of a chromatin code reader. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Development of genetically flexible mouse models of sarcoma using RCAS-TVA mediated gene delivery.

    Directory of Open Access Journals (Sweden)

    Leah Kabaroff

    Full Text Available Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively. Disrupting the G1 checkpoint CDKN2 (p16/p19-/- resulted in sarcoma in 30% of p16/p19-/- xN-tva mice with a median latency of 23 weeks (range 8-40 weeks. A similar incidence occurred in p16/p19-/- xBKE mice (32%, however, a shorter median latency (10.4 weeks was observed. p16/p19-/- xAKE mice also developed sarcomas (24% incidence; median 9 weeks yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.

  12. Disruption of Axonal Transport in Motor Neuron Diseases

    Directory of Open Access Journals (Sweden)

    Gen Sobue

    2012-01-01

    Full Text Available Motor neurons typically have very long axons, and fine-tuning axonal transport is crucial for their survival. The obstruction of axonal transport is gaining attention as a cause of neuronal dysfunction in a variety of neurodegenerative motor neuron diseases. Depletions in dynein and dynactin-1, motor molecules regulating axonal trafficking, disrupt axonal transport in flies, and mutations in their genes cause motor neuron degeneration in humans and rodents. Axonal transport defects are among the early molecular events leading to neurodegeneration in mouse models of amyotrophic lateral sclerosis (ALS. Gene expression profiles indicate that dynactin-1 mRNA is downregulated in degenerating spinal motor neurons of autopsied patients with sporadic ALS. Dynactin-1 mRNA is also reduced in the affected neurons of a mouse model of spinal and bulbar muscular atrophy, a motor neuron disease caused by triplet CAG repeat expansion in the gene encoding the androgen receptor. Pathogenic androgen receptor proteins also inhibit kinesin-1 microtubule-binding activity and disrupt anterograde axonal transport by activating c-Jun N-terminal kinase. Disruption of axonal transport also underlies the pathogenesis of spinal muscular atrophy and hereditary spastic paraplegias. These observations suggest that the impairment of axonal transport is a key event in the pathological processes of motor neuron degeneration and an important target of therapy development for motor neuron diseases.

  13. Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

    Science.gov (United States)

    Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

    2016-12-01

    Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

  14. Magnetic Resonance Imaging and Volumetric Analysis: Novel Tools to Study Thyroid Hormone Disruption and Its Effect on White Matter Development

    Science.gov (United States)

    Humans and wildlife are exposed to environmental pollutants that have been shown to interfere with the thyroid hormone system and thus may affect brain development. Our goal was to expose pregnant rats to propylthiouracil (PTU) to measure the effects of a goitrogen on white matte...

  15. Technology development for gene discovery and full-length sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Marcelo Bento Soares

    2004-07-19

    In previous years, with support from the U.S. Department of Energy, we developed methods for construction of normalized and subtracted cDNA libraries, and constructed hundreds of high-quality libraries for production of Expressed Sequence Tags (ESTs). Our clones were made widely available to the scientific community through the IMAGE Consortium, and millions of ESTs were produced from our libraries either by collaborators or by our own sequencing laboratory at the University of Iowa. During this grant period, we focused on (1) the development of a method for preferential cloning of tissue-specific and/or rare transcripts, (2) its utilization to expedite EST-based gene discovery for the NIH Mouse Brain Molecular Anatomy Project, (3) further development and optimization of a method for construction of full-length-enriched cDNA libraries, and (4) modification of a plasmid vector to maximize efficiency of full-length cDNA sequencing by the transposon-mediated approach. It is noteworthy that the technology developed for preferential cloning of rare mRNAs enabled identification of over 2,000 mouse transcripts differentially expressed in the hippocampus. In addition, the method that we optimized for construction of full-length-enriched cDNA libraries was successfully utilized for the production of approximately fifty libraries from the developing mouse nervous system, from which over 2,500 full-ORF-containing cDNAs have been identified and accurately sequenced in their entirety either by our group or by the NIH-Mammalian Gene Collection Program Sequencing Team.

  16. Disruptive Co-Creation?

    DEFF Research Database (Denmark)

    Butler, Ivan; Christensen, David

    set up to foster innovative partnerships between civil society organizations, businesses and academia with the objective of supporting disruptive business model innovation in co-creation processes across these sectors. This paper delves into state-of-the-art concerning literature pertinent......Faced with constant changes in the marketplace and increasing pressure to adapt to e.g. recessive and saturated conditions, businesses in industrialized countries have the option to pursue opportunities in emerging markets. In the poorest of developing countries, there is a proposition of high......-payoff, albeit high-risk opportunities for sustainable business models that address poverty alleviation. However, many businesses exhibit hesitation and wariness before giving serious consideration toward entering markets in e.g. Sub-Saharan Africa. In addressing this, the initiative access2innovation has been...

  17. Disruption of Radiologist Workflow.

    Science.gov (United States)

    Kansagra, Akash P; Liu, Kevin; Yu, John-Paul J

    2016-01-01

    The effect of disruptions has been studied extensively in surgery and emergency medicine, and a number of solutions-such as preoperative checklists-have been implemented to enforce the integrity of critical safety-related workflows. Disruptions of the highly complex and cognitively demanding workflow of modern clinical radiology have only recently attracted attention as a potential safety hazard. In this article, we describe the variety of disruptions that arise in the reading room environment, review approaches that other specialties have taken to mitigate workflow disruption, and suggest possible solutions for workflow improvement in radiology. Copyright © 2015 Mosby, Inc. All rights reserved.

  18. Non-trisomic homeobox gene expression during craniofacial development in the Ts65Dn mouse model of Down syndrome.

    Science.gov (United States)

    Billingsley, Cherie N; Allen, Jared R; Baumann, Douglas D; Deitz, Samantha L; Blazek, Joshua D; Newbauer, Abby; Darrah, Andrew; Long, Brad C; Young, Brandon; Clement, Mark; Doerge, R W; Roper, Randall J

    2013-08-01

    Trisomy 21 in humans causes cognitive impairment, craniofacial dysmorphology, and heart defects collectively referred to as Down syndrome. Yet, the pathophysiology of these phenotypes is not well understood. Craniofacial alterations may lead to complications in breathing, eating, and communication. Ts65Dn mice exhibit craniofacial alterations that model Down syndrome including a small mandible. We show that Ts65Dn embryos at 13.5 days gestation (E13.5) have a smaller mandibular precursor but a normal sized tongue as compared to euploid embryos, suggesting a relative instead of actual macroglossia originates during development. Neurological tissues were also altered in E13.5 trisomic embryos. Our array analysis found 155 differentially expressed non-trisomic genes in the trisomic E13.5 mandible, including 20 genes containing a homeobox DNA binding domain. Additionally, Sox9, important in skeletal formation and cell proliferation, was upregulated in Ts65Dn mandible precursors. Our results suggest trisomy causes altered expression of non-trisomic genes in development leading to structural changes associated with DS. Identification of genetic pathways disrupted by trisomy is an important step in proposing rational therapies at relevant time points to ameliorate craniofacial abnormalities in DS and other congenital disorders. Copyright © 2013 Wiley Periodicals, Inc.

  19. Gene network analysis in plant development by genomic technologies.

    Science.gov (United States)

    Wellmer, Frank; Riechmann, José Luis

    2005-01-01

    The analysis of the gene regulatory networks underlying development is of central importance for a better understanding of the mechanisms that control the formation of the different cell-types, tissues or organs of an organism. The recent invention of genomic technologies has opened the possibility of studying these networks at a global level. In this paper, we summarize some of the recent advances that have been made in the understanding of plant development by the application of genomic technologies. We focus on a few specific processes, namely flower and root development and the control of the cell cycle, but we also highlight landmark studies in other areas that opened new avenues of experimentation or analysis. We describe the methods and the strategies that are currently used for the analysis of plant development by genomic technologies, as well as some of the problems and limitations that hamper their application. Since many genomic technologies and concepts were first developed and tested in organisms other than plants, we make reference to work in non-plant species and compare the current state of network analysis in plants to that in other multicellular organisms.

  20. EFFECTS OF CHRONIC CADMIUM EXPOSURE ON METAMORPHOSIS, SKELETAL DEVELOPMENT AND THYROID ENDOCRINE DISRUPTION IN CHINESE TOAD BUFO GARGARIZANS TADPOLES.

    Science.gov (United States)

    Sun, Nailiang; Wang, Hongyuan; Ju, Zongqi; Zhao, Hongfeng

    2017-08-11

    The study examined the effects of chronic cadmium exposure on metamorphosis, body size, thyroid gland, and skeletal development of Chinese toad (Bufo gargarizans) tadpoles. Tadpoles were exposed to cadmium concentrations at 0, 5, 10, 50,100 and 500 μg/L from Gonser stage 26 to Gosner stage 46 of completion of metamorphosis. Our results showed 100 and 500 μg/L cadmium concentrations increased mortality and decelerated metamorphosis rate. In addition, significant body size reduction at Gosner stage 42 was observed at 100 and 500 μg/L cadmium treatments (p Bufo gargarizans larvae. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. 2005 Trophoblast Research Award Lecture: Defects in the keratin cytoskeleton disrupt normal murine placental development and trophoblast cell function.

    Science.gov (United States)

    Watson, E D

    2007-04-01

    The keratin cytoskeleton is present in all trophoblast cell subtypes of the mouse and human placenta and is required to maintain the structural integrity of these cells. Recently, various genetic mouse models have shown that a normal keratin network is necessary for placental development. Keratin-deficiency leads to trophoblast giant cell fragility, breaking the barrier between the conceptus and the maternal blood circulation. Alternatively, keratin aggregation prevents chorioallantoic attachment, a key developmental milestone required for the formation of the labyrinth within the mouse placenta. These models give us insight into cytokeratin function in human trophoblast cell subtypes and suggest that defects in the keratin cytoskeleton may result in intrauterine growth restriction or miscarriage.

  2. Potential complications when developing gene deletion clones in Xylella fastidiosa.

    Science.gov (United States)

    Johnson, Kameka L; Cursino, Luciana; Athinuwat, Dusit; Burr, Thomas J; Mowery, Patricia

    2015-04-16

    The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers. Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations. We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present. We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

  3. Prenatal brain disruption in molybdenum cofactor deficiency.

    Science.gov (United States)

    Carmi-Nawi, Nirit; Malinger, Gustavo; Mandel, Hanna; Ichida, Kimiyoshi; Lerman-Sagie, Tally; Lev, Dorit

    2011-04-01

    Molybdenum cofactor deficiency is a rare autosomal recessive disorder that may present during the neonatal period with intractable seizures and be mistaken for ischemic encephalopathy. We describe a patient whose prenatal sonography at 35 weeks' gestation revealed diffuse brain damage with multiple subcortical cavities, ventriculomegaly, dysgenesis of the corpus callosum, and a hypoplastic cerebellum with an enlarged cisterna magna. Magnetic resonance imaging (MRI) later revealed brain atrophy, and multicystic encephalomalacia with hypoplastic vermis and cerebellum. Neurological examination at 10 months showed microcephaly, profound mental retardation, and spasticity. Uric acid was low, and taurine and xanthine were increased in the urine. A sulfite test was positive. The diagnosis of molybdenum cofactor deficiency was made. Sulfite oxidase activity in fibroblasts was undetectable. The patient was found to be homozygous for the 251-418del in the MOCS1 gene. This is the first description of the prenatal development of severe brain disruption in molybdenum cofactor deficiency.

  4. Differential Gene Expression Patterns in Developing Sexually Dimorphic Rat Brain Regions Exposed to Antiandrogenic, Estrogenic, or Complex Endocrine

    DEFF Research Database (Denmark)

    Lichtensteiger, Walter; Bassetti-Gaille, Catherine; Faass, Oliver

    2015-01-01

    The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect...... to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E...... area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact...

  5. Effects of alachlor on the early development and induction of estrogen-responsive genes in Medaka, Oryzias latipes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, C.; Ryu, J.; Park, S.Y.; Choi, K.; Jeon, S.H.; Na, J.G.; Rhee, D.G. [National Inst. of Environmental Research, Incheon (Korea)

    2004-09-15

    Alachlor is an acetanilide herbicide used to control annual grasses and weeds in field corn, soybeans, and peanuts. It is a selective systemic herbicide, absorbed by germinating shoots and by roots. Although the specific pathways are not exactly understood, the acetanilide herbicides apparently interfere with several physiological processes including biosynthesis of lipids, proteins and flavonoids. These herbicides are widely used in agriculture and are commonly detected in surface water and groundwater. Alachlor has a relatively low acute toxicity, however, repeated exposure has been reported to cause hepatotoxicity, irreversible uveal degeneration and tumour formation in some animals. Besides alachlor is one of the herbicides reported to have endocrine disrupting effects. 2,4-D, 2,4,5-T, amitrole and atrazine also belong to these types of herbicides. Alachlor is a strongly suspected endocrine disruptor in that it is listed by EPA and the World Wildlife Fund [WWF] as a potential endocrine disrupting chemical. Many mammalian and aquatic toxicological studies with alachlor were performed under the conditions of acute, subacute and chronic experiment. However, not many studies using fish have been carried out with the purpose of screening and testing of endocrine disrupting effects of alachlor. The purpose of this study was to determine the effects of alachlor on the early morphological development of medaka (Oryzias latipes). Embryonic growth, deformation and hatching success were determined to see the effects of this chemical. Also, we tried to measure the estrogenic activity of alachlor using the ELISA and RT-PCR methods. By using these techniques, we evaluated the induction of the estrogen-responsive genes, vitellogenin (precursor of yolk protein) and choriogenin (precursor of egg envelope protein) in male medaka exposed to alachlor.

  6. Anterior Hox Genes in Cardiac Development and Great Artery Patterning

    Directory of Open Access Journals (Sweden)

    Brigitte Laforest

    2014-03-01

    Full Text Available During early development, the heart tube grows by progressive addition of progenitor cells to the arterial and venous poles. These cardiac progenitor cells, originally identified in 2001, are located in the splanchnic mesoderm in a region termed the second heart field (SHF. Since its discovery, our view of heart development has been refined and it is well established that perturbation in the addition of SHF cells results in a spectrum of congenital heart defects. We have previously shown that anterior Hox genes, including Hoxb1, Hoxa1 and Hoxa3, are expressed in distinct subdomains of the SHF that contribute to atrial and subpulmonary myocardium. It is well known that Hox proteins exert their function through interaction with members of the TALE family, including Pbx and Meis factors. The expression profile of Pbx and Meis factors overlaps with that of anterior Hox factors in the embryonic heart, and recent data suggest that they may interact together during cardiac development. This review aims to bring together recent findings in vertebrates that strongly suggest an important function for Hox, Pbx and Meis factors in heart development and disease.

  7. Unique spatiotemporal requirements for intraflagellar transport genes during forebrain development.

    Directory of Open Access Journals (Sweden)

    John Snedeker

    Full Text Available Primary cilia are organelles extended from virtually all cells and are required for the proper regulation of a number of canonical developmental pathways. The role in cortical development of proteins important for ciliary form and function is a relatively understudied area. Here we have taken a genetic approach to define the role in forebrain development of three intraflagellar transport proteins known to be important for primary cilia function. We have genetically ablated Kif3a, Ift88, and Ttc21b in a series of specific spatiotemporal domains. The resulting phenotypes allow us to draw several conclusions. First, we conclude that the Ttc21b cortical phenotype is not due to the activity of Ttc21b within the brain itself. Secondly, some of the most striking phenotypes are from ablations in the neural crest cells and the adjacent surface ectoderm indicating that cilia transduce critical tissue-tissue interactions in the developing embryonic head. Finally, we note striking differences in phenotypes from ablations only one embryonic day apart, indicating very discrete spatiotemporal requirements for these three genes in cortical development.

  8. Targeted disruption in mice of a neural stem cell-maintaining, KRAB-Zn finger-encoding gene that has rapidly evolved in the human lineage.

    Directory of Open Access Journals (Sweden)

    Huan-Chieh Chien

    Full Text Available Understanding the genetic basis of the physical and behavioral traits that separate humans from other primates is a challenging but intriguing topic. The adaptive functions of the expansion and/or reduction in human brain size have long been explored. From a brain transcriptome project we have identified a KRAB-Zn finger protein-encoding gene (M003-A06 that has rapidly evolved since the human-chimpanzee separation. Quantitative RT-PCR analysis of different human tissues indicates that M003-A06 expression is enriched in the human fetal brain in addition to the fetal heart. Furthermore, analysis with use of immunofluorescence staining, neurosphere culturing and Western blotting indicates that the mouse ortholog of M003-A06, Zfp568, is expressed mainly in the embryonic stem (ES cells and fetal as well as adult neural stem cells (NSCs. Conditional gene knockout experiments in mice demonstrates that Zfp568 is both an NSC maintaining- and a brain size-regulating gene. Significantly, molecular genetic analyses show that human M003-A06 consists of 2 equilibrated allelic types, H and C, one of which (H is human-specific. Combined contemporary genotyping and database mining have revealed interesting genetic associations between the different genotypes of M003-A06 and the human head sizes. We propose that M003-A06 is likely one of the genes contributing to the uniqueness of the human brain in comparison to other higher primates.

  9. Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

    OpenAIRE

    Malvar, T; Baum, J A

    1994-01-01

    The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.

  10. Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain.

    Directory of Open Access Journals (Sweden)

    Kevin D Broad

    Full Text Available In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

  11. Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster.

    Science.gov (United States)

    Abolaji, Amos O; Kamdem, Jean P; Lugokenski, Thiago H; Farombi, Ebenezer O; Souza, Diogo O; da Silva Loreto, Élgion L; Rocha, João B T

    2015-08-01

    The compounds 4-vinylcyclohexene 1,2-monoepoxide (VCM) and 4-Vinylcyclohexene diepoxide (VCD) are the two downstream metabolites of 4-vinylcyclohexene (VCH), an ovotoxic agent in mammals. In addition, VCM and VCD may be found as by-products of VCH oxidation in the environment. Recently, we reported the involvement of oxidative stress in the toxicity of VCH in Drosophila melanogaster. However, it was not possible to determine the individual contributions of VCM and VCD in VCH toxicity. Hence, we investigated the toxicity of VCM and VCD (10-1000 µM) in flies after 5 days of exposure via the diet. Our results indicated impairments in climbing behaviour and disruptions in antioxidant balance and redox status evidenced by an increase in DCFH oxidation, decreases in total thiol content and glutathione-S-transferase (GST) activity in the flies exposed to VCM and VCD (pmelanogaster has provided further lessons on the toxicity of VCM and VCD which suggest that the reported toxicity of VCH may be mediated by its transformation to VCM and VCD. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

    Directory of Open Access Journals (Sweden)

    Angela Cassese

    Full Text Available Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15 causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1. Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4 restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15 by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

  13. URBANIZATION AND ENVIRONMENTAL DISRUPTION OF CATCHMENT BASINS AS A RESULT OF HOUSING DEVELOPMENT AT CIDADE OPERARIA AND ADJACENCIES, MARANHAO ISLAND

    Directory of Open Access Journals (Sweden)

    Izis Deise Silva de Sousa

    2005-05-01

    Full Text Available The Industrial Revolution was followed by an increase in the rates of urbanization, particularlyafter the second-half of the 20th century. In this context, Sao Luis, the capital city ofMaranhao State, adopted a housing development policy that was aimed at reducing thehousing deficit in the municipality. The construction of the Cidade Operaria housing complexwas a response to these explicit social needs, which have intensified since the 1970s. Theoccupation of the complex, however, resulted in considerable damage to the ecologicalintegrity of the adjacent habitats. The complex is located in the catchment basin of thePaciencia and Santo Antonio rivers, at the center of the Maranhao Island. This study analyzesthe spatial and temporal dynamics of the area’s urbanization process, which occurred in threephases: the period between 1976 and 1981 saw the removal of the original vegetation cover insome areas, as a result of the first settlement in Parque Zelandia. During that period,subsistence agriculture was common practice along the margins of streams and rivers. Thesecond phase corresponds to the construction of the Cidade Operaria housing complex, whichoccurred between 1981 and 1988. During that time, 860 ha of land were cleared andurbanized, resulting in considerable environmental damage. Changes of relief, due to gradingand earthmoving activities, lead to irreversible modifications in the course of rivers andstreams; many tributaries of the rivers Paciencia and Santo Antonio had theirs springssmothered by earthfills; construction of houses and the use of asphalt on roads reduced soilpermeability, increasing the depth of the water table; the release of untreated sewagecontaminated the drainage basins. During the third phase, which started in 1988, theenvironmental degradation was intensified by the occupation of areas adjacent to the housingcomplex. This model of urbanization has caused irreversible damage to the hydrology andwater quality in the

  14. Disrupted Sleep: From Molecules to Cognition

    Science.gov (United States)

    Cirelli, Chiara; Dijk, Derk-Jan; Van Cauter, Eve; Schwartz, Sophie; Chee, Michael W.L.

    2015-01-01

    Although the functions of sleep remain to be fully elucidated, it is clear that there are far-reaching effects of its disruption, whether by curtailment for a single night, by a few hours each night over a long period, or by disruption in sleep continuity. Epidemiological and experimental studies of these different forms of sleep disruption show deranged physiology from subcellular levels to complex affective behavior. In keeping with the multifaceted influence of sleep on health and well-being, we illustrate how the duration of sleep, its timing, and continuity can affect cellular ultrastructure, gene expression, metabolic and hormone regulation, mood, and vigilance. Recent brain imaging studies provide some clues on mechanisms underlying the most common cause of disrupted sleep (insomnia). These insights should ultimately result in adequate interventions to prevent and treat sleep disruption because of their high relevance to our most prevalent health problems. SIGNIFICANCE STATEMENT Disruption of the duration, timing, and continuity of sleep affects cellular ultrastructure, gene expression, appetite regulation, hormone production, vigilance, and reward functions. PMID:26468189

  15. Airline Disruption Management - Perspectives, Experiences and Outlook

    DEFF Research Database (Denmark)

    Kohl, Niklas; Larsen, Allan; Larsen, Jesper

    2004-01-01

    by for example severe weather, technical problems and crew sickness. Thus, the field of Airline Disruption Management has emerged within the past few years. The increased focus on cutting cost at the major airlines has intensified the interest in the development of new and cost e cient methods to handle airline...... disruptions. The purpose of this paper is twofold. In the first part it o ers an introduction to airline disruption management, provides the readers with a description of the planning processes and delivers a detailed overview of the numerous aspects of airline disruption management. In the second part we...... report on experiences from a large research and development project on airline disruption management. Within the project the first prototype of a multiple resource decision support system at the operations control center in a major airline, has been implemented....

  16. Airline Disruption Management - Perspectives, Experiences and Outlook

    DEFF Research Database (Denmark)

    Kohl, Niklas; Larsen, Allan; Larsen, Jesper

    2007-01-01

    by for example severe weather, technical problems and crew sickness. Thus, the field of Airline Disruption Management has emerged within the past few years. The increased focus on cutting cost at the major airlines has intensified the interest in the development of new and cost efficient methods to handle...... airline disruptions. The purpose of this paper is twofold. In the first part it offers an introduction to airline disruption management provides the readers with a description of the planning processes and delivers a detailed overview of the numerous aspects of airline disruption management. In the second...... part we report on experiences from a large research and development project on airline disruption management. Within the project the first prototype of a multiple resource decision support system at the operations control center in a major airline, has been implemented....

  17. The paf gene product modulates asexual development in Penicillium chrysogenum.

    Science.gov (United States)

    Hegedüs, Nikoletta; Sigl, Claudia; Zadra, Ivo; Pócsi, Istvan; Marx, Florentine

    2011-06-01

    Penicillium chrysogenum secretes a low molecular weight, cationic and cysteine-rich protein (PAF). It has growth inhibitory activity against the model organism Aspergillus nidulans and numerous zoo- and phytopathogenic fungi but shows only minimal conditional antifungal activity against the producing organism itself. In this study we provide evidence for an additional function of PAF which is distinct from the antifungal activity against putative ecologically concurrent microorganisms. Our data indicate that PAF enhances conidiation in P. chrysogenum by modulating the expression of brlA, the central regulatory gene for mitospore development. A paf deletion strain showed a significant impairment of mitospore formation which sustains our hypothesis that PAF plays an important role in balancing asexual differentiation in P. chrysogenum. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Candidate gene approach identifies multiple genes and signaling pathways downstream of Tbx4 in the developing allantois.

    Directory of Open Access Journals (Sweden)

    Ripla Arora

    Full Text Available Loss of Tbx4 results in absence of chorio-allantoic fusion and failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. We reviewed the literature for genes implicated in chorio-allantoic fusion, cavitation and vascular plexus formation, processes affected in Tbx4 mutant allantoises. Using this candidate gene approach, we identified a number of genes downstream of Tbx4 in the allantois including extracellular matrix molecules Vcan, Has2, and Itgα5, transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2. In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and, in wildtype allantoises, an inhibitor of the canonical Wnt signaling pathway disrupts vascular plexus formation. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation.

  19. Gene therapies development: slow progress and promising prospect

    OpenAIRE

    Hanna, Eve; R?muzat, C?cile; Auquier, Pascal; Toumi, Mondher

    2017-01-01

    ABSTRACT Background: In 1989, the concept of human gene therapies has emerged with the first approved human gene therapy trial of Rosenberg et al. Gene therapies are considered as promising therapies applicable to a broad range of diseases. Objective: The objective of this study was to review the descriptive data on gene therapy clinical trials conducted worldwide between 1989 and 2015, and to discuss potential success rates of these trials over time and anticipated market launch in the upcom...

  20. Dissecting insect development : baculovirus-mediated gene silencing in insects

    NARCIS (Netherlands)

    Hajós, J.P.; Vermunt, A.W.M.; Zuidema, D.; Kulcsár, P.; Varjas, L.; Kort, de C.A.D.; Závodszky, P.; Vlak, J.M.

    1999-01-01

    A novel concept applying baculovirus-mediated gene silencing to study insect gene function and regulation is described in this paper. A recombinant baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), was constructed with the juvenile hormone esterase (JHE) gene from the

  1. Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field.

    Science.gov (United States)

    Liu, June; Cattadori, Isabella M; Sim, Derek G; Eden, John-Sebastian; Holmes, Edward C; Read, Andrew F; Kerr, Peter J

    2017-10-15

    The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined.IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the

  2. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

    Science.gov (United States)

    2011-01-01

    Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase

  3. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

    Directory of Open Access Journals (Sweden)

    Pakula Tiina

    2011-05-01

    Full Text Available Abstract Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and