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Sample records for cell igd deletion

  1. B Cell IgD Deletion Prevents Alveolar Bone Loss Following Murine Oral Infection

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    Pamela J. Baker

    2009-01-01

    and CD4+ T cells in immune normal mice compared to IgD deficient mice. These data suggest that IgD is an important mediator of alveolar bone resorption, possibly through antigen-specific coactivation of B cells and CD4+ T cells.

  2. IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

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    Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank

    2018-01-01

    Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626

  3. Interstitial Granulomatous Dermatitis (IGD

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    Tiberiu Tebeica

    2017-07-01

    Full Text Available We report the case of a 42 years old male patient suffering from skin changes , which appeared in the last 7-8 years.  Two biopsies were performed during the evolution of the lesion. Both showed similar findings that consisted in a busy dermis with interstitial, superficial and deep infiltrates of lymphocytes and histiocytes dispersed among collagen bundles, with variable numbers of neutrophils scattered throughout. Some histiocytes were clustered in poorly formed granuloma that included rare giant cells, with discrete Palisades and piecemeal collagen degeneration, but without mucin deposition or frank necrobiosis of collagen. The clinical and histologic findings were supportive for interstitial granulomatous dermatitis. Interstitial granulomatous dermatitis (IGD is a poorly understood entity that was regarded by many as belonging to the same spectrum of disease or even synonym with palisaded and neutrophilic granulomatous dermatitis (PNGD. Although IGD and PNGD were usually related to connective tissue disease, mostly rheumatoid arthritis, some patients with typical histologic findings of IGD never develop autoimmune disorders, but they have different underlying conditions, such as metabolic diseases, lymphoproliferative disorders or other malignant tumours. These observations indicate that IGD and PNGD are different disorders with similar manifestations.

  4. IgD in the reptile leopard gecko.

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    Gambón-Deza, Francisco; Espinel, Christian Sánchez

    2008-07-01

    Immunoglobulin D (IgD) has been a mysterious antibody ever since it was discovered in mammals 40 years ago. It shares with IgM the role of antigen-receptor in the membrane of mature B cells. The absence of IgD in birds and its description in bony fishes contributed to the confusion about its evolutionary origins. Recent studies have established the presence of IgD in the amphibian Xenopus tropicalis. It is essential to study IgD genes in reptiles in order to better understand the evolution of this immunoglobulin in vertebrates. We describe in this report the IgM and IgD genes of the reptile Eublepharis macularius. The IgM gene has characteristics that are similar to those described in other species whereas IgD gene departs from the normal structure described for this antibody class in other species. It is made up of 11 immunoglobulins domains without evidence of recent intragenic duplications of exons as described in IgD genes of fish and X.tropicalis. It is possible that the immunoglobulin is comprised of domains inherited from earlier species and that this form of IgD is close to that present in animals that left the sea to live on land. Furthermore, domains CH7 and CH8 of E. macularius IgD are orthologues to domains CH2 and CH3 of mammalian IgD. The present study also describes a second IgD (IgD2) which must have appeared recently by duplication of an older immunoglobulin gene and recombination with the IgA-like gene described in this specie. Tissue expression of IgD and IgD2 mRNA is similar to that of IgM mRNA, suggesting a functional role of reptilian IgD.

  5. Ku80-deleted cells are defective at base excision repair

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    Li, Han; Marple, Teresa; Hasty, Paul

    2013-01-01

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H 2 O 2 and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs

  6. Ku80-deleted cells are defective at base excision repair

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    Li, Han [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain); Marple, Teresa [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain)

    2013-05-15

    Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H{sub 2}O{sub 2} and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

  7. IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice

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    Choi, Jin Huk; Wang, Kuan-wen; Zhang, Duanwu; Zhan, Xiaowei; Wang, Tao; Bu, Chun-Hui; Behrendt, Cassie L.; Zeng, Ming; Wang, Ying; Misawa, Takuma; Li, Xiaohong; Tang, Miao; Zhan, Xiaoming; Scott, Lindsay; Hildebrand, Sara; Murray, Anne R.; Moresco, Eva Marie Y.; Hooper, Lora V.; Beutler, Bruce

    2017-01-01

    Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR. PMID:28137874

  8. IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice.

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    Choi, Jin Huk; Wang, Kuan-Wen; Zhang, Duanwu; Zhan, Xiaowei; Wang, Tao; Bu, Chun-Hui; Behrendt, Cassie L; Zeng, Ming; Wang, Ying; Misawa, Takuma; Li, Xiaohong; Tang, Miao; Zhan, Xiaoming; Scott, Lindsay; Hildebrand, Sara; Murray, Anne R; Moresco, Eva Marie Y; Hooper, Lora V; Beutler, Bruce

    2017-02-14

    Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR.

  9. Role of DNA deletion length in mutation and cell survival

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    Braby, L.A.; Morgan, T.L.

    1992-01-01

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  10. Determination of serum IgD radioimmunoassay

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    Fayol, V.; Hartmann, D.J.; Sabbagh, I.; Ville, G.

    1986-01-01

    We describe a sensitive liquid phase radioimmunoassay for serum IgD. Extreme values obtained from 85 control patients sera are 0.2 and 121 mg/l with an arithmetic mean of 25 mg/l. In atopic patients (with high serum IgE levels), arithmetic mean is 47 mg/l [fr

  11. Determination of serum IgD radioimmunoassay

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    Fayol, V.; Hartmann, D.J.; Sabbagh, I.; Ville, G.

    1986-01-01

    We describe a sensitive liquid phase radioimmunoassay for serum IgD. Extreme values obtained from 85 control patients sera are 0.2 and 121 mg/l with an arithmetic mean of 25 mg/l. In atopic patients (with high serum IgE levels), arithmetic mean is 47 mg/l.

  12. Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

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    Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

    2013-01-01

    In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

  13. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

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    Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J; Paton, Lois; Woof, Jenny M; von Pawel-Rammingen, Ulrich

    2016-01-01

    Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

  14. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

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    Lushaj, Entela B.; Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi

    2012-01-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  15. Rag Deletion in Peripheral T Cells Blocks TCR Revision

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    Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

    2010-01-01

    Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

  16. The first report of myeloma with IgD κ and free κ in Indonesia

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    Riadi Wirawan

    2011-08-01

    Full Text Available IgD κ myeloma is a rare plasma cell neoplasm case and has never been reported before in Indonesia. In normal condition, IgD level in blood is very low, therefore increase of IgD level in myeloma could be missed by serum protein electrophoresis. A case of a 59 years old female with severe bone pain is reported. In radiology evaluation, there were thoracal compression fracture and thoracal foramen narrowing. For this patient, the myeloma diagnosis was based on WHO criteria, the stage IIIb was based on Durie and Salmon criteria, and bad prognosis with prognostic index stage III diagnosis was based on International Prognostic Index from International Myeloma Working Group, respectively. In serum protein electrophoresis we found a very small monoclonal spike and in immunofi xation there were monoclonal IgD κ and free light chain κ. (Med J Indones 2011; 20:217-21Keywords: immunofixation, myeloma IgD κ, protein electrophoresis

  17. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

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    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee

    2009-01-01

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with 137 Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H 2 O 2 -treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H 2 O 2 -treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells

  18. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

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    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2009-06-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with {sup 137}Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H{sub 2}O{sub 2}-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H{sub 2}O{sub 2}-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

  19. Conditional IL-2 gene deletion: consequences for T cell proliferation

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    Kendall A Smith

    2012-05-01

    Full Text Available To explore the role of interleukin-2 (IL-2 in T cell proliferation, and to circumvent the IL-2 deficiency autoimmune syndrome of conventional il2 gene deletion, mice were created to allow conditional il2 gene deletion when treated with the estrogen analogue, tamoxifen (TAM as adults. Splenocytes from four different mouse strains, C57Bl/6 wild type (WT, conventional IL-2 (-/-, TAM-treated Cre recombinase negative (Cre-/IL2fl/fl, and Cre+/IL-2fl/fl (Cre+, were activated with anti-CD3 and anti-CD28, and monitored for CD4+ and CD8+ T cell lymphocyte blastogenesis, aerobic glycolysis, BrdU incorporation into newly synthesized DNA, and CFSE dye dilution to monitor cell division. IL-2 production was monitored by quantitative ELISA and multiple additional cytokines were monitored by protein-bead arrays. Splenocytes from conventional IL-2 (-/- and TAM-treated Cre+ mice resulted in undetectable IL-2 production, so that both strains were IL-2 deficient. As monitored by flow cytometry, activated CD4+ and CD8+ T cells from WT, Cre+ and Cre- mice all underwent blastogenesis, whereas far fewer cells from conventional IL-2 (-/- mice did so. By comparison, only cells from IL-2 sufficient WT and Cre- switched to aerobic glycolysis as evidenced by a drop in media pH. Blastogenesis was mirrored by BrdU incorporation and CFSE dye dilution by CD4+ and CD8+ T cells from WT, Cre+ and Cre- mice, which were all equivalent, while proliferation of cells from conventional IL-2 (-/- mice was compromised. Splenocytes from IL-2 deficient conventional IL-2 (-/- mice produced low or undetectable other γc-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21, whereas production of these γc-chain cytokines from IL-2-deficient conditional IL-2 (-/- Cre+ mice were comparable with WT and Cre- mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch to aerobic glycolysis is attributable to IL-2, and proliferation

  20. Detection of mitochondrial DNA deletions in human cells induced by ionizing radiation

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    Liu, Qing-Jie; Feng, Jiang-Bin; Lu, Xue; Li, Yu-Wen; Chen, De-Qing

    2008-01-01

    Full text: Purpose: To screen the novel mitochondrial DNA (mt DNA) deletions induced by ionizing radiation, and analyze the several kinds of mt DNA deletions, known as 3895 bp, 889 bp, 7436 bp or 4934 bp deletions. Methods: Long-range PCR with two pairs of primers, which could amplify the whole human mitochondrial genome, was used to analyze the lymphoblastoid cell line before and after exposed to 10 Gy 60 Co γ-rays. The limited condition PCR was used to certify the possible mt DNA deletion showed by long-range PCR. The PCR products were purified, cloned, sequenced and the sequence result were BLASTed. Regular PCR or nest-PCR were used to analyze the 3895 bp, 889 bp, 7436 bp or 4934 bp deletions before and after radiation exposure. The final PCR products were purified, sequenced and BALSTed on standard human mitochondrial genome sequence database. Results: (1) The predicted bands of mt DNA were observed on the control cell lines, and the possible mt DNA deletions were also detected on the irradiated cell lines. The deletions were certified by the limited condition PCR. The sequence BLAST results of the cloned PCR products showed that two kinds of deletions, 7455 bp deletion (nt 475-7929 in heavy strand) and 9225 bp deletion (nt 7714-369 in heavy strand), which were between two 8 bp direct repeats. Further bioinformatics analysis showed that the two deletions were novel deletions. (2) The 889 bp and 3895 bp deletion were not detected for the cell line samples not exposed to 60 Co γ-rays. The 889 bp and 3895 bp deletions were detected on samples exposed to 10 Gy 60 Co γ-rays. The BALST results showed that the 889 bp and 3895 deletions flanked nt 11688 bp-12576, nt 548 bp-4443, respectively. The 7436 bp deletion levels were not changed much before and after irradiation. (3) The 4934 bp deletions had the same pattern as 7436 bp deletion, but it could induced by radiation. Conclusions: Ionizing radiation could induce the human lymphoblastoid two novel mt DNA

  1. Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines

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    Nelson, S.L.; Grosovsky, A.J.; Jones, I.M.; Burkhart-Schultz, K.; Fuscoe, J.C.

    1995-01-01

    We have examined the extent of of HPRT - total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT - deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (>1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT - total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT - large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions. 24 refs., 6 figs., 1 tab

  2. Endothelial Plasmalemma Vesicle-Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells.

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    Elgueta, Raul; Tse, Dan; Deharvengt, Sophie J; Luciano, Marcus R; Carriere, Catherine; Noelle, Randolph J; Stan, Radu V

    2016-11-15

    Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM + IgD lo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM + IgD lo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM + IgD lo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM + B cells in the spleen and peritoneal cavity. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Adolescents with Internet Gaming Disorder (IGD): profiles and treatment response.

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    Martín-Fernández, María; Matalí, Josep Lluís; García-Sánchez, Sara; Pardo, Marta; Lleras, María; Castellano-Tejedor, Carmina

    2016-10-07

    Demand for treatment for problems related to the use of video games have increased significantly in adolescents. Most cases have a comorbid mental disorder that jeopardises both pathologies. The aim of this study is to describe profiles of adolescents with Internet Gaming Disorder (IGD) according to comorbidity and analyze treatment response at 3 and 6 months. A sample of 86 patients which consulted in the Addictive Behavior Unit of a hospital was assessed with diagnostic criteria for IGD, the interview K-SADS-PL for mental disorders and the Clinical Global Impression (CGI) to treatment progress. Of the initial sample, 68,6% (n = 59) met diagnostic criteria for IGD. Of these, the 45,76% matched an internalizing profile, presenting comorbidity with Mood Disorders (44,4%), Anxiety Disorders (44,4%) and Personality Disorders (11,1%). The externalizing profile would comprise 52,54% of the sample presenting Disruptive Behavior Disorder (48,4%=, ADHD (29%) and Disruptive Behavior Disorders not otherwise specified (22,6%). Unlike externalizing, the internalizing patients had a family history of psychiatric problems (63%), difficulties in social relationships (77,8%) and seemed to use video games preferably to escape discomfort (66,7%). After 3 months the externalizing profile showed improvements. Comorbid disorders allow the discrimination of two IGD profiles in adolescents and these could influence treatment response. Therefore, it is important to assess comorbidities to design a more accurate intervention focused on the specificities of each profile.

  4. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

    Science.gov (United States)

    LaSarge, Candi L; Santos, Victor R; Danzer, Steve C

    2015-03-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Prediction of radiosensitivity of human tumor cell lines in vitro by determining 4977bp deletion in mitochondrial DNA

    International Nuclear Information System (INIS)

    Rong Qinglin; Cao Yongzhen; Zhang Yaowen; Zhao Xinran; Wang Qin; Li Jin; Liu Qiang

    2008-01-01

    Objective: To evaluate the possibility of predicting the radiosensitivity of tumor cell lines using the assay of the mtDNA4977bp deletion. Methods: The mtDNA4977bp deletion of HepG 2 cells and PC-3 cells were detected by nested PCR after irradiated by various doses of x-ray. Results: The radiation-induced mtDNA4977bp deletion of the tumor cell lines of HepG 2 and PC-3 were detected after irradiated. There was a dose dependent in the mtDNA4977bp deletion of two tumor cell lines. The deletion rate of HepG 2 was higher significantly than that of PC-3 at each point of radiation dose (P 2 was higher than that of PC-3. Conclusion: The assay of the mtDNA4977bp deletion may be an approach to predict the radiosensitivity of tumor cells. (authors)

  6. Prognostic impact of IKZF1 deletion in adults with common B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Yao, Qiu-Mei; Liu, Kai-Yan; Gale, Robert Peter; Jiang, Bin; Liu, Yan-Rong; Jiang, Qian; Jiang, Hao; Zhang, Xiao-Hui; Zhang, Mei-Jie; Chen, Shan-Shan; Huang, Xiao-Jun; Xu, Lan-Ping; Ruan, Guo-Rui

    2016-04-11

    Interrogate the impact of IKZF1 deletion on therapy-outcomes of adults with common B-cell acute lymphoblastic leukemia. One hundred sixty-five consecutive adults with common B-cell ALL were tested for IKZF1 deletion and for BCR/ABL. Deletions in IKZF1 were detected using multiplex RQ-PCR, multiplex fluorescent PCR, sequence analysis and multiplex ligation-dependent probe amplification (MLPA). BCR/ABL was detected using RQ-PCR. All subjects received chemotherapy and some also received an allotransplant and tyrosine kinase-inhibitors. Multivariate analyses were done to identify associations between IKZF1 deletion and other variables on non-relapse mortality (NRM), cumulative incidence of relapse (CIR), leukemia-free survival (LFS) and survival. Amongst subjects achieving complete remission those with IKZF1 deletion had similar 5-year non-relapse mortality (NRM) (11% [2-20%] vs. 16% [4-28%]; P = 0.736), a higher 5-year cumulative incidence of relapse (CIR) (55% [35-76%] vs. 25% [12-38%]; P = 0.004), and worse 5-year leukemia-free survival (LFS) (33% [16-52%] vs. 59% [42-73%]; P = 0.012) and survival (48% [33-62%] vs. 75% [57-86%]; P = 0.002). In multivariate analyses IKZF1 deletion was associated with an increased relapse (relative risk [RR] =2.7, [1.4-5.2]; P = 0.002), a higher risk of treatment-failure (inverse of LFS; RR = 2.1, [1.2-3.6]; P = 0.007) and a higher risk of death (RR = 2.8, [1.5-5.5]; P = 0.002). The adverse impact of IKZF1 deletion on outcomes was stronger in subjects without vs. with BCR-ABL1 and in subjects receiving chemotherapy-only vs. an allotransplant. IKZF1 deletion was independently-associated with a higher relapse risk and worse LFS and survival in adults with common B-cell ALL after adjusting for other prognostic variables and differences in therapies. These data suggest IKZF1 deletion may be a useful prognostic variable in adults with common B-cell ALL, especially in persons without BCR-ABL1 and those receiving chemotherapy

  7. Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

    Science.gov (United States)

    Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

    2009-01-16

    Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

  8. IgD, cyclooxygenase-2 and ribosomal protein S6-PS240 immune response in a case of early psoriasis

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2018-02-01

    Full Text Available Psoriasis is an inflammatory skin disease. Five classic types of psoriasis have been defined: plaque, inverse, pustular, guttate, and erythrodermic. The early psoriasis immunologic skin immune response is not well understood. Here we aim to show an immune and cell signaling response in a case of early psoriasis. A 56 year old female presented with a desquamative lesion on her right leg. A skin biopsy for hematoxylin and eosin (H&E and immunohistochemistry (IHC staining was taken. The diagnosis indicated early psoriasis, and IHC showed positive IgD staining in the epidermal corneal layer, as well as positive staining with ribosomal protein S6-pS240 (RIBO in the hyperproliferative epidermis. Cyclooxygenase-2 (COX-2 was also very positive in the granular layer in spots, at the basement membrane zone of the skin and in the inflammatory infiltrate in the dermis subjacent to hyperproliferative psoriatic areas. In an early case of psoriasis, we confirmed the presence of IgD, RIBO and COX-2. Each molecule seems to be playing a role in inflammation and intracellular signaling pathways in early psoriasis. The role of IgD is unknown, and this case brings to light the complexity of the pathologic changes occurring in early psoriatic lesions.

  9. IKZF1 DELETIONS ARE INDEPENDENT PROGNOSTIC FACTOR IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    G. A. Tsaur

    2016-01-01

    Full Text Available We assessed the prognostic significance of IKZF1 gene deletions in 141 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL  on Russian multicenter trial in pediatric clinics of Ekaterinburg and Orenburg. IKZF1 deletions were estimated by multiplex ligation-dependent probe amplification. IKZF1 deletions were revealed in 15 (10.6 % patients. IKZF1 deletions were associated with age older than 10 years (p = 0.007, initial white blood cell count higher than 30 × 109/l (p = 0.003, t(9;22(q34.q11 (p = 0.003 and delayed blast clearance: М3 status of bone marrow at day 15 of remission induction (p = 0.003, lack of hematological remission at day 36 (p < 0.001 and high levels of minimal residual disease at days 15, 36 and 85 (p = 0.014; p < 0.001; p = 0.001 correspondingly. Patients with IKZF1 deletions had significantly lower event-free survival (EFS (0.30 ± 0.15 vs 0.89 ± 0.03; p < 0.001 and overall survival (OS (0.44 ± 0.19 vs 0.93 ± 0.02; p < 0.001, while cumulative incidence of relapse was higher (0.67 ± 0.18 vs 0.07 ± 0.02; p < 0.001. In the multivariate analysis IKZF1 deletions were associated with decreased EFS (hazard ratio (HR 4.755; 95 % confidence interval (CI 1.856–12.185; p = 0.001, and OS (HR 4.208; 95 % CI 1.322–13.393; p = 0.015, but increased relapse risk (HR 9,083; 95 % CI 3.119–26.451; p < 0.001. IKZF1 deletions retained their prognostic significance in the intermediate risk group patients (p < 0.001, but not in standard or high-risk groups. Majority of IKZF1 deletions – 12 (80 % of 15 – were revealed in the “B-other” group (n = 83. In this cohort of patients IKZF1 deletions led to inferior EFS (HR 6.172; 95 % CI 1.834–20.767; p = 0.003 and higher relapse rate (HR 16.303; 95 % CI 3.324–79.965; p = 0.015. Thus, our results showed that IKZF1 deletions are independent risk factor in BCP-ALL patients.

  10. Allelic deletions of cell growth regulators during progression of bladder cancer

    DEFF Research Database (Denmark)

    Primdahl, H; von der Maase, H; Christensen, M

    2000-01-01

    Cell growth regulators include proteins of the p53 pathway encoded by the genes CDKN2A (p16, p14arf), MDM2, TP53, and CDKN1A (p21) as well as proteins encoded by genes like RB1, E2F, and MYCL. In the present study we investigated allelic deletions of all these genes in each recurrent bladder tumor...... difference in the numbers of gene loci hit by deletions muscle-invasive versus noninvasive tumors (P = 0.0000002), with the genes most often hit by deletions in muscle-invasive tumors being TP53, RB1, and MYCL. A number of novel findings were made. Losses of MYCL and RB1 alleles were more pronounced...... that a characteristic difference between recurrent noninvasive and recurrent progressing bladder tumors is loss of cell cycle-regulatory genes in the latter group....

  11. Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-14-1-0251 TITLE: Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase... Tyrosine Kinase Inhibitors 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0251 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kiran Mahajan 5d...ABSTRACT Central to all cycling cells-including prostate cancer stem cells- is the expression of WEE1 tyrosine kinase. WEE1 monitors duplication of

  12. Radiosensitivity evaluation of Human tumor cell lines by detecting 4977bp deletion in mitochondrial DNA

    International Nuclear Information System (INIS)

    Zhang Yipei

    2009-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA4977bp deletion. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA4977bp deletion and DNA damage were detected by MTT assay and nested PCR technique respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4and 8Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. PCR method:The differences on mtDNA 4977bp deletion in mitochondrial DNA among HepG 2 , EC-9706 and MCF-7 were not significant after 1Gy and 4Gy γ-ray irradiation. The ratio of 4977bp deletion in mitochondrial DNA of HepG 2 and EC-9706 increased while that of MCF-7 decreased after 8Gy irradiation. The ratio of mtDNA 4977bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7, which implies that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF -7. Conclusion: As a new biological marker, mtDNA4977bp deletion may be hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  13. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; de Boer, J.G.; de Jong, P.J.; Drobetsky, E.A.; Glickman, B.W.

    1988-01-01

    The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage

  14. Prevalence of glutathione S-transferase gene deletions and their effect on sickle cell patients

    Directory of Open Access Journals (Sweden)

    Pandey Sanjay

    2012-01-01

    Full Text Available BACKGROUND: Glutathione S-transferase gene deletions are known detoxification agents and cause oxidative damage. Due to the different pathophysiology of anemia in thalassemia and sickle cell disease, there are significant differences in the pathophysiology of iron overload and iron-related complications in these disorders. OBJECTIVE: The aim of this study was to estimate the frequency of the GSTM1 and GSTT1 genotypes in sickle cell disease patients and their effect on iron status. METHODS: Forty sickle cell anemia and sixty sickle ß-thalassemia patients and 100 controls were evaluated to determine the frequency of GST gene deletions. Complete blood counts were performed by an automated cell analyzer. Hemoglobin F, hemoglobin A, hemoglobin A2 and hemoglobin S were measured and diagnosis of patients was achieved by high performance liquid chromatography with DNA extraction by the phenol-chloroform method. The GST null genotype was determined using multiplex polymerase chain reaction and serum ferritin was measured using an ELISA kit. Statistical analysis was by EpiInfo and GraphPad statistics software. RESULTS: An increased frequency of the GSTT1 null genotype (p-value = 0.05 was seen in the patients. The mean serum ferritin level was higher in patients with the GST genotypes than in controls; this was statistically significant for all genotypes except GSTM1, however the higher levels of serum ferritin were due to blood transfusions in patients. CONCLUSION: GST deletions do not play a direct role in iron overload of sickle cell patients.

  15. Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus

    International Nuclear Information System (INIS)

    Papadopoulo, D.; Guillouf, C.; Moustacchi, E.; Mohrenweiser, H.

    1990-01-01

    Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing of DNA cross-links and certain monoadducts. The authors reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture of cross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus they propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves by passing lesions and subsequent gap filling by a recombinational process during replication

  16. SNX27 Deletion Causes Hydrocephalus by Impairing Ependymal Cell Differentiation and Ciliogenesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Ying; Wang, Jian; Tseng, I-Chu; Huang, Timothy; Zhao, Yingjun; Zheng, Qiuyang; Gao, Yue; Luo, Hong; Zhang, Xian; Bu, Guojun; Hong, Wanjin; Xu, Huaxi

    2016-12-14

    Hydrocephalus is a brain disorder derived from CSF accumulation due to defects in CSF clearance. Although dysfunctional apical cilia in the ependymal cell layer are causal to the onset of hydrocephalus, mechanisms underlying proper ependymal cell differentiation are largely unclear. SNX27 is a trafficking component required for normal brain function and was shown previously to suppress γ-secretase-dependent amyloid precursor protein and Notch cleavage. However, it was unclear how SNX27-dependent γ-secretase inhibition could contribute to brain development and pathophysiology. Here, we describe and characterize an Snx27-deleted mouse model for the ependymal layer defects of deciliation and hydrocephalus. SNX27 deficiency results in reductions in ependymal cells and cilia density, as well as severe postnatal hydrocephalus. Inhibition of Notch intracellular domain signaling with γ-secretase inhibitors reversed ependymal cells/cilia loss and dilation of lateral ventricles in Snx27-deficient mice, giving strong indication that Snx27 deletion triggers defects in ependymal layer formation and ciliogenesis through Notch hyperactivation. Together, these results suggest that SNX27 is essential for ependymal cell differentiation and ciliogenesis, and its deletion can promote hydrocephalus pathogenesis. Down's syndrome (DS) in humans and mouse models has been shown previously to confer a high risk for the development of pathological hydrocephalus. Because we have previously described SNX27 as a component that is consistently downregulated in DS, we present here a robust Snx27-deleted mouse model that produces hydrocephalus and associated ciliary defects with complete penetrance. In addition, we find that γ-secretase/Notch modulation may be a candidate drug target in SNX27-associated hydrocephalus such as that observed in DS. Based on these findings, we anticipate that future study will determine whether modulation of a SNX27/Notch/γ-secretase pathway can also be of

  17. IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice

    OpenAIRE

    Choi, Jin Huk; Wang, Kuan-wen; Zhang, Duanwu; Zhan, Xiaowei; Wang, Tao; Bu, Chun-Hui; Behrendt, Cassie L.; Zeng, Ming; Wang, Ying; Misawa, Takuma; Li, Xiaohong; Tang, Miao; Zhan, Xiaoming; Scott, Lindsay; Hildebrand, Sara

    2017-01-01

    Immunoglobulins exist in several forms, or isotypes, that carry out distinct effector functions. During an antibody response, B cells can switch their immunoglobulin isotype through the process of class-switch recombination (CSR). CSR to IgD is a rare event compared with CSR to other isotypes, and its regulation is poorly understood. Here we report that mice lacking the DNA damage-response protein 53BP1 display a hyper-IgD syndrome despite deficiencies of other immunoglobulin classes. By stud...

  18. Deletions of the long arm of chromosome 5 define subgroups of T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    La Starza, Roberta; Barba, Gianluca; Demeyer, Sofie; Pierini, Valentina; Di Giacomo, Danika; Gianfelici, Valentina; Schwab, Claire; Matteucci, Caterina; Vicente, Carmen; Cools, Jan; Messina, Monica; Crescenzi, Barbara; Chiaretti, Sabina; Foà, Robin; Basso, Giuseppe; Harrison, Christine J; Mecucci, Cristina

    2016-08-01

    Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia. Copyright© Ferrata Storti Foundation.

  19. Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

    Science.gov (United States)

    Völkening, Bianca; Schönig, Kai; Kronenberg, Golo; Bartsch, Dusan; Weber, Tillmann

    2017-05-01

    Ca 2+ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Ca v 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Ca v 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created Tg GLAST-CreERT2 /Cacna1c fl/fl /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Ca v 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Ca v 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Ca v 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion. © 2017 Wiley Periodicals, Inc.

  20. Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

    Science.gov (United States)

    Muñoz, Jorge; Lázcoz, Paula; Inda, María Mar; Nistal, Manuel; Pestaña, Angel; Encío, Ignacio J; Castresana, Javier S

    2004-05-01

    Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing. Copyright 2004 Wiley-Liss, Inc.

  1. Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

    Directory of Open Access Journals (Sweden)

    Konstantin V Salojin

    Full Text Available Mammalian sterile 20-like kinase 1 (Mst1 is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

  2. Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

    Science.gov (United States)

    Nagashima, Kazuki; Sawa, Shinichiro; Nitta, Takeshi; Prados, Alejandro; Koliaraki, Vasiliki; Kollias, George; Nakashima, Tomoki; Takayanagi, Hiroshi

    2017-11-04

    The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin + CD31 - cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin + CD31 - cells. Tnfsf11 fl/Δ Col6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Clinical implications of cytosine deletion of exon 5 of P53 gene in non small cell lung cancer patients

    Directory of Open Access Journals (Sweden)

    Rashid Mir

    2016-01-01

    Full Text Available Aim: Lung cancer is considered to be the most common cancer in the world. In humans, about 50% or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate the cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Our study was aimed to evaluate the frequency of cytosine deletion in nonsmall cell lung cancer (NSCLC patients. Methods: One hundred NSCLC patients were genotyped for P53 (exon5, codon168 cytosine deletion leading to loss of its function and activate the target genes by allele-specific polymerase chain reaction. The P53 cytosine deletion was correlated with all the clinicopathological parameters of the patients. Results and Analysis: 59% cases were carrying P53 cytosine deletion. Similarly, the significantly higher incidence of cytosine deletion was reported in current smokers (75% in comparison to exsmoker and nonsmoker. Significantly higher frequency of cytosine deletion was reported in adenocarcinoma (68.08% than squamous cell carcinoma (52.83%. Also, a significant difference was reported between p53 cytosine deletion and metastasis (64.28%. Further, the majority of the cases assessed for response carrying P53 cytosine deletion were found to show faster disease progression. Conclusion: The data suggests that there is a significant association of the P53 exon 5 deletion of cytosine in codon 168 with metastasis and staging of the disease.

  4. A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Y.; Li, X.M.; Shapiro, L.J. [UCSF School of Medicine, San Francisco, CA (United States)] [and others

    1994-09-01

    Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand, and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.

  5. Genetic deletion of Rnd3 in neural stem cells promotes proliferation via upregulation of Notch signaling.

    Science.gov (United States)

    Dong, Huimin; Lin, Xi; Li, Yuntao; Hu, Ronghua; Xu, Yang; Guo, Xiaojie; La, Qiong; Wang, Shun; Fang, Congcong; Guo, Junli; Li, Qi; Mao, Shanping; Liu, Baohui

    2017-10-31

    Rnd3, a Rho GTPase, is involved in the inhibition of actin cytoskeleton dynamics through the Rho kinase-dependent signaling pathway. We previously demonstrated that mice with genetic deletion of Rnd3 developed a markedly larger brain compared with wild-type mice. Here, we demonstrate that Rnd3 knockout mice developed an enlarged subventricular zone, and we identify a novel role for Rnd3 as an inhibitor of Notch signaling in neural stem cells. Rnd3 deficiency, both in vivo and in vitro , resulted in increased levels of Notch intracellular domain protein. This led to enhanced Notch signaling and promotion of aberrant neural stem cell growth, thereby resulting in a larger subventricular zone and a markedly larger brain. Inhibition of Notch activity abrogated this aberrant neural stem cell growth.

  6. Increased IgD milk antibody responses in a patient with Down's syndrome, pulmonary hemosiderosis and cor pulmonale.

    Science.gov (United States)

    Galant, S; Nussbaum, E; Wittner, R; DeWeck, A L; Heiner, D C

    1983-10-01

    IgD antibody responses to cow's milk were investigated in a two-year-old black boy with evidence of pulmonary hemosiderosis and pulmonary hypertension. Initially a broad spectrum of immunologic responses to cow's milk were observed including IgD, IgE, and precipitin antibodies. Specific IgD antibody responses to cow's milk could be modulated in terms of challenge or elimination and correlated with the clinical course. It is possible that IgD antibodies may be important in milk-related pulmonary hemosiderosis.

  7. Conditional deletion of Cadherin 13 perturbs Golgi cells and disrupts social and cognitive behaviors.

    Science.gov (United States)

    Tantra, M; Guo, L; Kim, J; Zainolabidin, N; Eulenburg, V; Augustine, G J; Chen, A I

    2018-02-15

    Inhibitory interneurons mediate the gating of synaptic transmission and modulate the activities of neural circuits. Disruption of the function of inhibitory networks in the forebrain is linked to impairment of social and cognitive behaviors, but the involvement of inhibitory interneurons in the cerebellum has not been assessed. We found that Cadherin 13 (Cdh13), a gene implicated in autism spectrum disorder and attention-deficit hyperactivity disorder, is specifically expressed in Golgi cells within the cerebellar cortex. To assess the function of Cdh13 and utilize the manipulation of Cdh13 expression in Golgi cells as an entry point to examine cerebellar-mediated function, we generated mice carrying Cdh13-floxed alleles and conditionally deleted Cdh13 with GlyT2::Cre mice. Loss of Cdh13 results in a decrease in the expression/localization of GAD67 and reduces spontaneous inhibitory postsynaptic current (IPSC) in cerebellar Golgi cells without disrupting spontaneous excitatory postsynaptic current (EPSC). At the behavioral level, loss of Cdh13 in the cerebellum, piriform cortex and endopiriform claustrum have no impact on gross motor coordination or general locomotor behaviors, but leads to deficits in cognitive and social abilities. Mice lacking Cdh13 exhibit reduced cognitive flexibility and loss of preference for contact region concomitant with increased reciprocal social interactions. Together, our findings show that Cdh13 is critical for inhibitory function of Golgi cells, and that GlyT2::Cre-mediated deletion of Cdh13 in non-executive centers of the brain, such as the cerebellum, may contribute to cognitive and social behavioral deficits linked to neurological disorders. © 2018 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.

  8. Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

    Science.gov (United States)

    Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

    2013-01-01

    In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

  9. Homozygous deletion of the α- and β1-interferon genes in human leukemia and derived cell lines

    International Nuclear Information System (INIS)

    Diaz, M.O.; Ziemin, S.; Le Beau, M.M.; Pitha, P.; Smith, S.D.; Chilcote, R.R.; Rowley, J.D.

    1988-01-01

    The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm [del(9p)], unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The α- and β 1 -interferon genes have been assigned to this chromosome region (9p21-22). The authors now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5'-methylthioadenosine phosphorylase, an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them

  10. Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

    Science.gov (United States)

    Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

    2014-01-01

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. PMID:24907273

  11. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

    Science.gov (United States)

    Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek; Yien, Yvette Y; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H; Orkin, Stuart H

    2014-08-01

    The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance

    Directory of Open Access Journals (Sweden)

    Dempwolff Felix

    2012-12-01

    Full Text Available Abstract Background In eukaryotic cells, dynamin and flotillin are involved in processes such as endocytosis and lipid raft formation, respectively. Dynamin is a GTPase that exerts motor-like activity during the pinching off of vesicles, while flotillins are coiled coil rich membrane proteins with no known enzymatic activity. Bacteria also possess orthologs of both classes of proteins, but their function has been unclear. Results We show that deletion of the single dynA or floT genes lead to no phenotype or a mild defect in septum formation in the case of the dynA gene, while dynA floT double mutant cells were highly elongated and irregularly shaped, although the MreB cytoskeleton appeared to be normal. DynA colocalizes with FtsZ, and the dynA deletion strain shows aberrant FtsZ rings in a subpopulation of cells. The mild division defect of the dynA deletion is exacerbated by an additional deletion in ezrA, which affects FtsZ ring formation, and also by the deletion of a late division gene (divIB, indicating that DynA affects several steps in cell division. DynA and mreB deletions generated a synthetic defect in cell shape maintenance, showing that MreB and DynA play non-epistatic functions in cell shape maintenance. TIRF microscopy revealed that FloT forms many dynamic membrane assemblies that frequently colocalize with the division septum. The deletion of dynA did not change the pattern of localization of FloT, and vice versa, showing that the two proteins play non redundant roles in a variety of cellular processes. Expression of dynamin or flotillin T in eukaryotic S2 cells revealed that both proteins assemble at the cell membrane. While FloT formed patch structures, DynA built up tubulated structures extending away from the cells. Conclusions Bacillus subtilis dynamin ortholog DynA plays a role during cell division and in cell shape maintenance. It shows a genetic link with flotillin T, with both proteins playing non-redundant functions at

  13. Stepping back to advance: Why IGD needs an intensified debate instead of a consensus.

    Science.gov (United States)

    Quandt, Thorsten

    2017-06-01

    Based on their analysis of Internet gaming disorder (IGD) criteria, Kuss, Griffiths, and Pontes (2017) come to the conclusion that the current situation can be described as "chaos and confusion." Their assessment is not an exaggeration. It can be argued that there are even more issues, on logical/definitional and political/social levels: (a) the IGD diagnosis is lacking a well-defined object, (b) the cause and effect cannot be differentiated outside lab conditions, (c) the social and political effects of declaring a social behavior as a disease are worrying, and (d) a rushed diagnosis may construct an addiction with potentially harmful effects on (formerly) healthy populations. Instead of closing the debate by declaring a consensus and codifying IGD in the DSM, an undogmatic, intensified, and broader discussion is needed.

  14. TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Mark A.; Shekhar, Tanmay M. [Department of Biochemistry and Genetics, La Trobe University, Bundoora, Victoria (Australia); La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia); Hall, Nathan E. [La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia); Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, Melbourne, Victoria (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry and Genetics, La Trobe University, Bundoora, Victoria (Australia); La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria (Australia)

    2016-05-15

    Highlights: • Treatment with TRAIL or EMS provokes mutations in clonogenically viable TK6 cells. • TRAIL is 2–5-fold less mutagenic than an equivalently lethal concentration of EMS. • EMS mainly causes transition mutations at the HPRT and TK1 loci of TK6 cells. • Most loss-of-function HPRT or TK1 mutations caused by TRAIL treatment are deletions. - Abstract: When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of

  15. TRAIL causes deletions at the HPRT and TK1 loci of clonogenically competent cells

    International Nuclear Information System (INIS)

    Miles, Mark A.; Shekhar, Tanmay M.; Hall, Nathan E.; Hawkins, Christine J.

    2016-01-01

    Highlights: • Treatment with TRAIL or EMS provokes mutations in clonogenically viable TK6 cells. • TRAIL is 2–5-fold less mutagenic than an equivalently lethal concentration of EMS. • EMS mainly causes transition mutations at the HPRT and TK1 loci of TK6 cells. • Most loss-of-function HPRT or TK1 mutations caused by TRAIL treatment are deletions. - Abstract: When chemotherapy and radiotherapy are effective, they function by inducing DNA damage in cancerous cells, which respond by undergoing apoptosis. Some adverse effects can result from collateral destruction of non-cancerous cells, via the same mechanism. Therapy-related cancers, a particularly serious adverse effect of anti-cancer treatments, develop due to oncogenic mutations created in non-cancerous cells by the DNA damaging therapies used to eliminate the original cancer. Physiologically achievable concentrations of direct apoptosis inducing anti-cancer drugs that target Bcl-2 and IAP proteins possess negligible mutagenic activity, however death receptor agonists like TRAIL/Apo2L can provoke mutations in surviving cells, probably via caspase-mediated activation of the nuclease CAD. In this study we compared the types of mutations sustained in the HPRT and TK1 loci of clonogenically competent cells following treatment with TRAIL or the alkylating agent ethyl methanesulfonate (EMS). As expected, the loss-of-function mutations in the HPRT or TK1 loci triggered by exposure to EMS were almost all transitions. In contrast, only a minority of the mutations identified in TRAIL-treated clones lacking HPRT or TK1 activity were substitutions. Almost three quarters of the TRAIL-induced mutations were partial or complete deletions of the HPRT or TK1 genes, consistent with sub-lethal TRAIL treatment provoking double strand breaks, which may be mis-repaired by non-homologous end joining (NHEJ). Mis-repair of double-strand breaks following exposure to chemotherapy drugs has been implicated in the pathogenesis of

  16. Conditional deletion of pejvakin in adult outer hair cells causes progressive hearing loss in mice.

    Science.gov (United States)

    Harris, Suzan L; Kazmierczak, Marcin; Pangršič, Tina; Shah, Prahar; Chuchvara, Nadiya; Barrantes-Freer, Alonso; Moser, Tobias; Schwander, Martin

    2017-03-06

    Mutations in the Pejvakin (Pjvk) gene cause autosomal recessive hearing loss DFNB59 with audiological features of auditory neuropathy spectrum disorder (ANSD) or cochlear dysfunction. The precise mechanisms underlying the variable clinical phenotypes of DFNB59 remain unclear. Here, we demonstrate that mice with conditional ablation of the Pjvk gene in all sensory hair cells or only in outer hair cells (OHCs) show similar auditory phenotypes with early-onset profound hearing loss. By contrast, loss of Pjvk in adult OHCs causes a slowly progressive hearing loss associated with OHC degeneration and delayed loss of inner hair cells (IHCs), indicating a primary role for pejvakin in regulating OHC function and survival. Consistent with this model, synaptic transmission at the IHC ribbon synapse is largely unaffected in sirtaki mice that carry a C-terminal deletion mutation in Pjvk. Using the C-terminal domain of pejvakin as bait, we identified in a cochlear cDNA library ROCK2, an effector for the small GTPase Rho, and the scaffold protein IQGAP1, involved in modulating actin dynamics. Both ROCK2 and IQGAP1 associate via their coiled-coil domains with pejvakin. We conclude that pejvakin is required to sustain OHC activity and survival in a cell-autonomous manner likely involving regulation of Rho signaling. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration.

    Science.gov (United States)

    Yan, Xiao; Himburg, Heather A; Pohl, Katherine; Quarmyne, Mamle; Tran, Evelyn; Zhang, Yurun; Fang, Tiancheng; Kan, Jenny; Chao, Nelson J; Zhao, Liman; Doan, Phuong L; Chute, John P

    2016-11-01

    Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10 m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10 +/+ mice. After total body irradiation (TBI), Grb10 m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10 +/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Biased Iglambda expression in hypermutated IgD multiple myelomas does not result from receptor revision

    NARCIS (Netherlands)

    van der Burg, M.; Bende, R. J.; Aarts, W. M.; Langerak, A. W.; van Dongen, J. J. M.; van Noesel, C. J. M.

    2002-01-01

    Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal

  19. The remnant of the European rabbit (Oryctolagus cuniculus IgD gene.

    Directory of Open Access Journals (Sweden)

    Dennis K Lanning

    Full Text Available Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (Cδ gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit Cδ gene approximately 21 kb downstream of the IgM gene. The remnant Cδ locus lacks the δCH1 and hinge exons, but contains truncated δCH2 and δCH3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the Cδ gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.

  20. Deletional rearrangement in the human T-cell receptor α-chain locus

    International Nuclear Information System (INIS)

    de Villartay, J.P.; Lewis, D.; Hockett, R.; Waldmann, T.A.; Korsmeyer, S.J.; Cohen, D.I.

    1987-01-01

    The antigen-specific receptor on the surface of mature T lymphocytes is a heterodimer consisting of polypeptides termed α and β. In the course of characterizing human T-cell tumors with an immature (CD4 - , CD8 - ) surface phenotype, the authors detected a 2-kilobase α-related transcript. Analysis of cDNA clones corresponding to this transcript established that a genetic element (which they call TEA, for T early α) located between the α-chain variable- and joining-region genes had been spliced to the α constant region. The TEA transcript is present early in thymocyte ontogeny, and its expression declines during T-cell maturation. More important, the TEA area functions as an active site for rearrangement within the α gene locus. Blot hybridization of restriction enzyme-digested DNA with a TEA probe revealed a narrowly limited pattern of rearrangement in polyclonal thymic DNA, surprisingly different from the pattern expected for the mature α gene with its complex diversity. These DNA blots also showed that TEA is generally present in the germ-line configuration in cells expressing the γδ heterodimeric receptor and is deleted from mature (αβ-expressing) T-lymphocyte tumors and lines. Moreover, the TEA transcript lacked a long open reading frame for protein but instead possessed multiple copies of a repetitive element resembling those utilized in the heavy-chain class switch of the immunoglobulin genes. The temporal nature of the rearrangements and expression detected by TEA suggests that this recombination could mediate a transition between immature (γδ-expressing) T cells and mature (αβ-expressing) T cells

  1. Fumarate Hydratase Deletion in Pancreatic β Cells Leads to Progressive Diabetes

    Directory of Open Access Journals (Sweden)

    Julie Adam

    2017-09-01

    Full Text Available We explored the role of the Krebs cycle enzyme fumarate hydratase (FH in glucose-stimulated insulin secretion (GSIS. Mice lacking Fh1 in pancreatic β cells (Fh1βKO mice appear normal for 6–8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1βKO mice led to dysregulated metabolism in β cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D. The impaired GSIS in islets from diabetic Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

  2. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  3. Conditional deletion of Hdac3 in osteoprogenitor cells attenuates diet-induced systemic metabolic dysfunction

    Science.gov (United States)

    McGee-Lawrence, Meghan E.; White, Thomas A.; LeBrasseur, Nathan K.; Westendorf, Jennifer J.

    2015-01-01

    Obesity is a major health epidemic in the United States and a leading cause of preventable diseases including type 2 diabetes. A growing body of evidence indicates that the skeleton influences whole body metabolism and suggests a new avenue for developing novel therapeutic agents, but the underlying mechanisms are not well understood. Here, it is demonstrated that conditional deletion of an epigenetic regulator, Hdac3, in osteoblast progenitor cells abrogates high fat diet-induced insulin resistance and hepatic steatosis. These Hdac3-deficient mice have reduced bone formation and lower circulating levels of total and undercarboxylated osteocalcin, coupled with decreased bone resorption activity. They also maintain lower body fat and fasting glucose levels on normal and high fat chow diets. The mechanisms by which Hdac3 controls systemic energy homeostasis from within osteoblasts have not yet been fully realized, but the current study suggests that it does not involve elevated levels of circulating osteocalcin. Thus, Hdac3 is a new player in the emerging paradigm that the skeleton influences systemic energy metabolism. PMID:25666992

  4. Radiosensitivity evaluation of human tumor cell lines by detecting 4977 bp deletion in mitochondrial DNA and comet assay

    International Nuclear Information System (INIS)

    Chu Liping; Liu Qiang; Wang Qin; Li Jin; Yue Jingyin; Mu Chuanjie; Fan Feiyue

    2008-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA 4977 bp deletion and comet assay. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA 4977 bp deletion and DNA damage were detected by MTY assay, nested PCR technique and comet assay, respectively. Results: The results of MTT assay showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. The ratio of mtDNA 4977 bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7 (P 2 and EC-9706 was higher than that of MCF-7. The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusions: Combination of many biological parameter is helpful to evaluate the radiosensitivity of tumor cells more accurately. (authors)

  5. Deletion of the calmodulin-binding domain of Grb7 impairs cell attachment to the extracellular matrix and migration

    Energy Technology Data Exchange (ETDEWEB)

    García-Palmero, Irene; Villalobo, Antonio, E-mail: antonio.villalobo@iib.uam.es

    2013-06-28

    Highlights: •Grb7 is a calmodulin (CaM)-binding protein. •Deleting the CaM-binding site impairs cell attachment and migration. •CaM antagonists inhibit Grb7-mediated cell migration. •We conclude that CaM controls Grb7-mediated cell migration. -- Abstract: The adaptor Grb7 is a calmodulin (CaM)-binding protein that participates in signaling pathways involved in cell migration, proliferation and the control of angiogenesis, and plays a significant role in tumor growth, its metastatic spread and tumor-associated neo-vasculature formation. In this report we show that deletion of the CaM-binding site of Grb7, located in the proximal region of its pleckstrin homology (PH) domain, impairs cell migration, cell attachment to the extracellular matrix, and the reorganization of the actin cytoskeleton occurring during this process. Moreover, we show that the cell-permeable CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) both retard the migration of cells expressing wild type Grb7, but not the migration of cells expressing the mutant protein lacking the CaM-binding site (Grb7Δ), underscoring the proactive role of CaM binding to Grb7 during this process.

  6. Ptpn11 Deletion in CD4+ Cells Does Not Affect T Cell Development and Functions but Causes Cartilage Tumors in a T Cell-Independent Manner.

    Science.gov (United States)

    Miah, S M Shahjahan; Jayasuriya, Chathuraka T; Salter, Alexander I; Reilly, Emma C; Fugere, Céline; Yang, Wentian; Chen, Qian; Brossay, Laurent

    2017-01-01

    The ubiquitously expressed tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2 (SHP-2, encoded by Ptpn11 ) is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, dephosphorylate key players of activating receptor signaling pathways. We hypothesized that SHP-2 deletion would have major effects on T cell development by altering the thresholds for activation, as well as positive and negative selection. Surprisingly, using mice conditionally deficient for SHP-2 in the T cell lineage, we show that the development of these lymphocytes is globally intact. In addition, our data demonstrate that SHP-2 absence does not compromise T cell effector functions, suggesting that SHP-2 is dispensable in these cells. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in wrist bones in a T cell-independent manner. These tumors resemble miniature cartilaginous growth plates and contain CD4-lineage positive chondrocyte-like cells. Altogether these results indicate that SHP-2 is a cartilage tumor suppressor during aging.

  7. Ptpn11 Deletion in CD4+ Cells Does Not Affect T Cell Development and Functions but Causes Cartilage Tumors in a T Cell-Independent Manner

    Directory of Open Access Journals (Sweden)

    S. M. Shahjahan Miah

    2017-10-01

    Full Text Available The ubiquitously expressed tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2 (SHP-2, encoded by Ptpn11 is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, dephosphorylate key players of activating receptor signaling pathways. We hypothesized that SHP-2 deletion would have major effects on T cell development by altering the thresholds for activation, as well as positive and negative selection. Surprisingly, using mice conditionally deficient for SHP-2 in the T cell lineage, we show that the development of these lymphocytes is globally intact. In addition, our data demonstrate that SHP-2 absence does not compromise T cell effector functions, suggesting that SHP-2 is dispensable in these cells. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in wrist bones in a T cell-independent manner. These tumors resemble miniature cartilaginous growth plates and contain CD4-lineage positive chondrocyte-like cells. Altogether these results indicate that SHP-2 is a cartilage tumor suppressor during aging.

  8. Deletion of PPAR-γ in immune cells enhances susceptibility to antiglomerular basement membrane disease

    Directory of Open Access Journals (Sweden)

    Cristen Chafin

    2010-10-01

    Full Text Available Cristen Chafin2, Sarah Muse2, Raquel Hontecillas5, Josep Bassaganya-Riera5, David L Caudell2, Samuel K Shimp III4, M Nichole Rylander4, John Zhang6, Liwu Li3, Christopher M Reilly1,21Virginia College of Osteopathic Medicine, 2Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 3Department of Biological Sciences, 4Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 5Nutritional Immunology and Molecular Medicine Laboratory, Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA; 6Medical University of SC, Charleston, SC, USAAbstract: Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-γ has been shown to be immunoregulatory in autoimmune diseases by inhibiting production of a number of inflammatory mediators. We investigated whether PPAR-γ gene deletion in hematopoietic cells would alter disease pathogenesis in the antiglomerular basement membrane (anti-GBM mouse model. PPAR-γ+/+ and PPAR-γ-/- mice were immunized with rabbit antimouse GBM antibodies and lipopolysaccharide and evaluated for two weeks. Although both the PPAR-γ+/+ and PPAR-γ-/- mice had IgG deposition in the glomerulus and showed proteinuria two weeks after injection, glomerular and tubulointerstitial disease in PPAR-γ-/- mice were significantly more severe compared with the PPAR-γ+/+ animals. We observed that the PPAR-γ-/- mice had decreased CD4+CD25+ regulatory T cells and an increased CD8+:CD4+ ratio as compared with the PPAR-γ+/+ mice, suggesting that PPAR-γ has a role in the regulation of T cells. Furthermore, plasma interleukin-6 levels were significantly increased in the PPAR-γ-/- mice at two weeks as compared with the PPAR-γ+/+ animals. Taken together, these studies show that

  9. IKZF1 deletion is associated with a poor outcome in pediatric B-cell precursor acute lymphoblastic leukemia in Japan

    International Nuclear Information System (INIS)

    Asai, Daisuke; Imamura, Toshihiko; Suenobu, So-ichi; Saito, Akiko; Hasegawa, Daiichiro; Deguchi, Takao; Hashii, Yoshiko; Matsumoto, Kimikazu; Kawasaki, Hirohide; Hori, Hiroki; Iguchi, Akihiro; Kosaka, Yoshiyuki; Kato, Koji; Horibe, Keizo; Yumura-Yagi, Keiko; Hara, Junichi; Oda, Megumi

    2013-01-01

    Genetic alterations of Ikaros family zinc finger protein 1 (IKZF1), point mutations in Janus kinase 2 (JAK2), and overexpression of cytokine receptor-like factor 2 (CRLF2) were recently reported to be associated with poor outcomes in pediatric B-cell precursor (BCP)-ALL. Herein, we conducted genetic analyses of IKZF1 deletion, point mutation of JAK2 exon 16, 17, and 21, CRLF2 expression, the presence of P2RY8-CRLF2 fusion and F232C mutation in CRLF2 in 202 pediatric BCP-ALL patients newly diagnosed and registered in Japan Childhood Leukemia Study ALL02 protocol to find out if alterations in these genes are determinants of poor outcome. All patients showed good response to initial prednisolone (PSL) treatment. Ph + , infantile, and Down syndrome–associated ALL were excluded. Deletion of IKZF1 occurred in 19/202 patients (9.4%) and CRLF2 overexpression occurred in 16/107 (15.0%), which are similar to previous reports. Patients with IKZF1 deletion had reduced event-free survival (EFS) and overall survival (OS) compared to those in patients without IKZF1 deletion (5-year EFS, 62.7% vs. 88.8%, 5-year OS, 71.8% vs. 90.2%). Our data also showed significantly inferior 5-year EFS (48.6% vs. 84.7%, log rank P = 0.0003) and 5-year OS (62.3% vs. 85.4%, log rank P = 0.009) in NCI-HR patients (n = 97). JAK2 mutations and P2RY8-CRLF2 fusion were rarely detected. IKZF1 deletion was identified as adverse prognostic factor even in pediatric BCP-ALL in NCI-HR showing good response to PSL

  10. Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

    Science.gov (United States)

    Pesko, Kendra N; Fitzpatrick, Kelly A; Ryan, Elizabeth M; Shi, Pei-Yong; Zhang, Bo; Lennon, Niall J; Newman, Ruchi M; Henn, Matthew R; Ebel, Gregory D

    2012-05-25

    Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. RCSD1-ABL1 Translocation Associated with IKZF1 Gene Deletion in B-Cell Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    Shawana Kamran

    2015-01-01

    Full Text Available The RCSD1 gene has recently been identified as a novel gene fusion partner of the ABL1 gene in cases of B-cell Acute Lymphoblastic Leukemia (B-ALL. The RCSD1 gene is located at 1q23 and ABL1 is located at 9q34, so that the RCSD1-ABL1 fusion typically arises through a rare reciprocal translocation t(1;9(q23;q34. Only a small number of RCSD1-ABL1 positive cases of B-ALL have been described in the literature, and the full spectrum of clinical, morphological, immunophenotypic, and molecular features associated with this genetic abnormality has not been defined. We describe extensive genetic characterization of a case of B-ALL with RCSD1-ABL1 fusion, by using conventional cytogenetic analysis, Fluorescence In Situ Hybridization (FISH studies, and Chromosomal Microarray Analysis (CMA. The use of CMA resulted in detection of an approximately 70 kb deletion at 7p12.2, which caused a disruption of the IKZF1 gene. Deletions and mutations of IKZF1 are recurring abnormalities in B-ALL and are associated with a poor prognosis. Our findings highlight the association of the deletion of IKZF1 gene with the t(1;9(q24;q34 and illustrate the importance of comprehensive cytogenetic and molecular evaluation for accurate prediction of prognosis in patients with B-cell ALL.

  12. Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

    Directory of Open Access Journals (Sweden)

    Danyi Peng

    2017-12-01

    Full Text Available Alveolar epithelial cells (AECs injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF. Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ model, as well as a new SMARCA4-deleted alveolar type II (ATII-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.

  13. Deletion of ALS5, ALS6 or ALS7 increases adhesion of Candida albicans to human vascular endothelial and buccal epithelial cells

    OpenAIRE

    ZHAO, XIAOMIN; OH, SOON-HWAN; HOYER, LOIS L.

    2007-01-01

    C. albicans yeast forms deleted for ALS5, ALS6 or ALS7 are more adherent than a relevant control strain to human vascular endothelial cell monolayers and buccal epithelial cells. In the buccal and vaginal reconstituted human epithelium (RHE) disease models, however, mutant and control strains caused a similar degree of tissue destruction. Deletion of ALS5 or ALS6 significantly slowed growth of the mutant strain; this phenotype was not affected by addition of excess uridine to the culture medi...

  14. PJA-BP expression and TCR delta deletion during human T cell differentiation

    NARCIS (Netherlands)

    M.C.M. Verschuren (Martie); B. Blom (Bianca); A.J.J.C. Bogers (Ad); H. Spits (Hergen); J.J.M. van Dongen (Jacques)

    1998-01-01

    textabstractRecombination of deltaRec to psiJalpha will delete the TCR delta gene, which is thought to play an important role in the bifurcation of the TCR alphabeta versus TCR gammadelta differentiation lineages. We recently detected a DNA-binding protein in human

  15. Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

    DEFF Research Database (Denmark)

    Kalis, Martins; Bolmeson, Caroline; Esguerra, Jonathan L.S.

    2011-01-01

    -cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre(+/-) Dicer1(¿/wt)), RIP-Cre(+/-)Dicer1(flox/flox) mice (RIP-Cre Dicer1(¿/¿)) developed progressive hyperglycaemia and full...... revealed altered islet morphology, marked decreased ß-cell mass, reduced numbers of granules within the ß-cells and reduced granule docking in adult RIP-Cre Dicer1(¿/¿) mice. ß-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal ß-cell development as 2-week old RIP-Cre Dicer1...

  16. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

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    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  17. Impaired Autophagy and Defective T Cell Homeostasis in Mice with T Cell-Specific Deletion of Receptor for Activated C Kinase 1

    Directory of Open Access Journals (Sweden)

    Guihua Qiu

    2017-05-01

    Full Text Available Autophagy plays a central role in maintaining T cell homeostasis. Our previous study has shown that hepatocyte-specific deficiency of receptor for activated C kinase 1 (RACK1 leads to lipid accumulation in the liver, accompanied by impaired autophagy, but its in vivo role in T cells remains unclear. Here, we report that mice with T cell-specific deletion of RACK1 exhibit normal intrathymic development of conventional T cells and regulatory T (Treg cells but reduced numbers of peripheral CD4+ and CD8+ T cells. Such defects are cell intrinsic with impaired mitochondrial clearance, increased sensitivity to cell death, and decreased proliferation that could be explained by impaired autophagy. Furthermore, RACK1 is essential for invariant natural T cell development. In vivo, T cell-specific loss of RACK1 dampens concanavalin A-induced acute liver injury. Our data suggest that RACK1 is a key regulator of T cell homeostasis.

  18. The Genetic Deletion of 6q21 and PRDM1 and Clinical Implications in Extranodal NK/T Cell Lymphoma, Nasal Type

    Directory of Open Access Journals (Sweden)

    Li Liang

    2015-01-01

    Full Text Available 6q21 genetic deletion has been frequently detected in extranodal NK/T cell lymphoma, nasal type (EN-NK/T-NT, and PRDM1 is considered as candidate gene. However, direct detection of PRDM1 deletion has not been well documented. We investigated genetic alterations of 6q21 and PRDM1 in 43 cases of EN-NK/T-NT and cell lines by FISH. PRDM1 expression was evaluated by immunohistochemistry and Western blot. The correlation between genetic alteration and PRDM1 expression and the significance in clinic-pathologic were analyzed. Heterozygous deletion of 6q21 and/or PRDM1 was observed in 24 of 43 cases (55.81% of EN-NK/T-NT including 16 cases (37.21% for 6q21 deletion and 19 cases (44.19% for PRDM1 deletion. Similarly, heterozygous codeletion of 6q21 and PRDM1 was identified in NK92 and NKL cells. The heterozygous deletion of 6q21 and/or PRDM1 was correlated with PRDM1 expression. However, genetic deletion of 6q21 and/or PRDM1 was not correlated with clinicopathological features of EN-NK/T-NT, while PRDM1 expression showed positive effect on the outcome of patients as those as disease site, B symptom, and clinical stage. Thus, heterozygous deletion of 6q21 and/or PRDM1 was frequently detected in EN-NK/T-NT and correlated with downregulation of PRDM1. But the prognostic role of genetic deletion needs to be further evaluated in larger cohort.

  19. Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

    Science.gov (United States)

    van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

    2017-06-15

    Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

  20. Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis

    DEFF Research Database (Denmark)

    Tanii, H; Ankarcrona, M; Flood, F

    2000-01-01

    . In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed...

  1. Sequential Kinase Inhibition (Idelalisib/Ibrutinib Induces Clinical Remission in B-Cell Prolymphocytic Leukemia Harboring a 17p Deletion

    Directory of Open Access Journals (Sweden)

    H. Coelho

    2017-01-01

    Full Text Available B-cell prolymphocytic leukemia (B-PLL is a rare lymphoid neoplasm with an aggressive clinical course. Treatment strategies for B-PLL remain to be established, and, until recently, alemtuzumab was the only effective therapeutic option in patients harboring 17p deletions. Herein, we describe, for the first time, a case of B-cell prolymphocytic leukemia harboring a 17p deletion in a 48-year-old man that was successfully treated sequentially with idelalisib-rituximab/ibrutinib followed by allogeneic hematopoietic stem cell transplant (allo-HSCT. After 5 months of therapy with idelalisib-rituximab, clinical remission was achieved, but the development of severe diarrhea led to its discontinuation. Subsequently, the patient was treated for 2 months with ibrutinib and the quality of the response was maintained with no severe adverse effects reported. A reduced-intensity conditioning allo-HSCT from a HLA-matched unrelated donor was performed, and, thereafter, the patient has been in complete remission for 10 months now. In conclusion, given the poor prognosis of B-PLL and the lack of effective treatment modalities, the findings here suggest that both ibrutinib and idelalisib should be considered as upfront therapy of B-PLL and as a bridge to allo-HSCT.

  2. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    Science.gov (United States)

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  3. [Distribution of abnormal cell clone with deletion of chromosome 20q in marrow cell lineages and apoptosis cells in myelodysplastic syndrome].

    Science.gov (United States)

    Qin, Ling; Wang, Chun; Qin, You-Wen; Xie, Kuang-Cheng; Yan, Shi-Ke; Gao, Yan-Rong; Wang, Xiao-Rui; Zhao, Chu-Xian

    2008-06-01

    This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

  4. IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly.

    Directory of Open Access Journals (Sweden)

    Lisa M Maurer

    Full Text Available Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K of fibronectin (FN stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD motifs in four of the nine FN type 1 (FNI modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3FNI and (5FNI; (7FNI and (9FNI; or (3FNI, (5FNI, (7FNI, and (9FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

  5. IGDS/TRAP Interface Program (ITIP). Software User Manual (SUM). [network flow diagrams for coal gasification studies

    Science.gov (United States)

    Jefferys, S.; Johnson, W.; Lewis, R.; Rich, R.

    1981-01-01

    This specification establishes the requirements, concepts, and preliminary design for a set of software known as the IGDS/TRAP Interface Program (ITIP). This software provides the capability to develop at an Interactive Graphics Design System (IGDS) design station process flow diagrams for use by the NASA Coal Gasification Task Team. In addition, ITIP will use the Data Management and Retrieval System (DMRS) to maintain a data base from which a properly formatted input file to the Time-Line and Resources Analysis Program (TRAP) can be extracted. This set of software will reside on the PDP-11/70 and will become the primary interface between the Coal Gasification Task Team and IGDS, DMRS, and TRAP. The user manual for the computer program is presented.

  6. Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations

    Directory of Open Access Journals (Sweden)

    Eva Severinson

    2017-05-01

    Full Text Available We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with interleukin-4 (IL-4 and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide that lack these cytoskeletal changes. We identified 84 genes with 10-fold or greater expression in anti-CD40 + IL-4 stimulated B cells, one of these encoded the guanine nucleotide exchange factor (GEF dedicator of cytokinesis 10 (Dock10. IL-4 selectively induced Dock10 expression in B cells. Using lacZ expression to monitor Dock10 promoter activity, we found that Dock10 was expressed at all stages during B cell development. However, specific deletion of Dock10 in B cells was associated with a mild phenotype with normal B cell development and normal B cell spreading, polarization, motility, chemotaxis, aggregation, and Ig class switching. Dock10-deficient B cells showed lower proliferation in response to anti-CD40 and IL-4 stimulation. Moreover, the IgG response to soluble antigen in vivo was lower when Dock10 was specifically deleted in B cells. Together, we found that most B cell responses were intact in the absence of Dock10. However, specific deletion of Dock10 in B cells was associated with a mild reduction in B cell activation in vitro and in vivo.

  7. Genetic deletion of afadin causes hydrocephalus by destruction of adherens junctions in radial glial and ependymal cells in the midbrain.

    Directory of Open Access Journals (Sweden)

    Hideaki Yamamoto

    Full Text Available Adherens junctions (AJs play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell-cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell-cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.

  8. IKAROS Gene Deleted B-Cell Acute Lymphoblastic Leukemia in Mexican Mestizos: Observations in Seven Patients and a Short Review of the Literature.

    Science.gov (United States)

    Ruiz-Delgado, Guillermo José; Cantero-Fortiz, Yahveth; León-Peña, Andrés Aurelio; León-González, Mónica; Nuñez-Cortés, Ana Karen; Ruiz-Argüelles, Guillermo José

    2016-01-01

    In B-cell acute lymphoblastic leukemia, one of the most frequent cytogenetic alterations is the presence of the Philadelphia chromosome. Recently, newly identified genetic alterations have been studied, among them the IKZF1 deletion. IKZF1 encodes IKAROS, a zinc finger protein that plays an important role in hematopoiesis involving the regulation process of adhesion, cellular migration, and as a tumor suppressor. We aimed to study the impact of IKAROS deletion in the evolution and prognosis of B-cell acute lymphoblastic leukemia. At a single center we prospectively studied patients diagnosed with B-cell acute lymphoblastic leukemia and screened for IKZF1 deletion using the multiplex ligation-dependent probe amplification method. We did a descriptive analysis of patients positive for the IKZF1 deletion to determine its impact on the evolution of the disease and survival rate. Between 2010 and 2015, 16 Mexican mestizo patients with B-cell acute lymphoblastic leukemia were prospectively screened for IKZF1 deletion; seven (43%) were positive and were included for further analysis. The age range of patients was 13-60 years; six were males and one female. All cases had type B acute lymphoblastic leukemia. Of the seven patients, two died, three were lost to follow-up, and two continue in complete remission with treatment. Results are worse than those in a group of patients with non-mutated IKAROS B-cell acute lymphoblastic leukemia previously studied in our center. Although this is a small sample, the presence of IKAROS deletion in acute lymphoblastic leukemia patients could represent a poor-prognosis marker and was probably related to therapy failure. It is also possible that this variant of leukemia may be more prevalent in Mexico. More studies are needed to define the role of IKZF1 deletion in acute lymphoblastic leukemia and the real prevalence of the disease in different populations.

  9. Binding of cetuximab to the EGFRvIII deletion mutant and its biological consequences in malignant glioma cells

    International Nuclear Information System (INIS)

    Jutten, Barry; Dubois, Ludwig; Li Younan; Aerts, Hugo; Wouters, Bradly G.; Lambin, Philippe; Theys, Jan; Lammering, Guido

    2009-01-01

    Background and purpose: Despite the clinical use of cetuximab, a chimeric antibody against EGFR, little is known regarding its interaction with EGFRvIII, a frequently expressed deletion mutant of EGFR. Therefore, we investigated the interaction and the functional consequences of cetuximab treatment on glioma cells stably expressing EGFRvIII. Materials and methods: The human glioma cell line U373 genetically modified to express EGFRvIII was used to measure the binding of cetuximab and its internalization using flow cytometry and confocal microscopy. Proliferation and cell survival were analyzed by cell growth and clonogenic survival assays. Results: Cetuximab is able to bind to EGFRvIII and causes an internalization of the receptor and decreases its expression levels. Furthermore, in contrast to EGF, cetuximab was able to activate EGFRvIII which was evidenced by multiple phosphorylation sites and its downstream signaling targets. Despite this activation, the growth rate and the radiosensitivity of the EGFRvIII-expressing glioma cells were not modulated. Conclusions: Cetuximab binds to EGFRvIII and leads to the initial activation, internalization and subsequent downregulation of EGFRvIII, but it does not seem to modulate the proliferation or radiosensitivity of EGFRvIII-expressing glioma cells. Thus, approaches to treat EGFRvIII-expressing glioma cells should be evaluated more carefully.

  10. Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis

    DEFF Research Database (Denmark)

    Carobbio, Stefania; Frigerio, Francesca; Rubi, Blanca

    2009-01-01

    Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been...... tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin...... secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored...

  11. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira

    2007-01-01

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53ΔC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53ΔC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain

  12. Deletion/duplication mutation screening of TP53 gene in patients with transitional cell carcinoma of urinary bladder using multiplex ligation-dependent probe amplification.

    Science.gov (United States)

    Bazrafshani, Mohammad Reza R; Nowshadi, Pouriaali A; Shirian, Sadegh; Daneshbod, Yahya; Nabipour, Fatemeh; Mokhtari, Maral; Hosseini, Fatemehsadat; Dehghan, Somayeh; Saeedzadeh, Abolfazl; Mosayebi, Ziba

    2016-02-01

    Bladder cancer is a molecular disease driven by the accumulation of genetic, epigenetic, and environmental factors. The aim of this study was to detect the deletions/duplication mutations in TP53 gene exons using multiplex ligation-dependent probe amplification (MLPA) method in the patients with transitional cell carcinoma (TCC). The achieved formalin-fixed paraffin-embedded tissues from 60 patients with TCC of bladder were screened for exonal deletions or duplications of every 12 TP53 gene exons using MLPA. The pathological sections were examined by three pathologists and categorized according to the WHO scoring guideline as 18 (30%) grade I, 22 (37%) grade II, 13 (22%) grade III, and 7 (11%) grade IV cases of TCC. None mutation changes of TP53 gene were detected in 24 (40%) of the patients. Furthermore, mutation changes including, 15 (25%) deletion, 17 (28%) duplication, and 4 (7%) both deletion and duplication cases were observed among 60 samples. From 12 exons of TP53 gene, exon 1 was more subjected to exonal deletion. Deletion of exon 1 of TP53 gene has occurred in 11 (35.4%) patients with TCC. In general, most mutations of TP53, either deletion or duplication, were found in exon 1, which was statistically significant. In addition, no relation between the TCC tumor grade and any type of mutation were observed in this research. MLPA is a simple and efficient method to analyze genomic deletions and duplications of all 12 exons of TP53 gene. The finding of this report that most of the mutations of TP53 occur in exon 1 is in contrast to that of the other reports suggesting that exons 5-8 are the most (frequently) mutated exons of TP53 gene. The mutations of exon 1 of TP53 gene may play an important role in the tumorogenesis of TCC. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  13. The influence of large deletions on the mutation frequency induced by tritiated water and X-radiation in male Drosophila melanogaster post-meiotic germ cells

    International Nuclear Information System (INIS)

    Fossett, N.G.; Byrne, B.J.; Kelley, S.J.; Tucker, A.B.; Arbour-Reily, P.; Lee, W.R.

    1994-01-01

    Tritium beta radiation ( 3 H β-radiation) in the form of tritiated water was used to induce mutations at the alcohol dehydrogenase (Adh) locus in male Drosophila melanogaster post-meiotic germ cells. All 23 Adh null mutations were large deletions (>20 kb), determined by genetic complementation and Southern blot analyses. 27 Adh null mutations have been induced by 100-kVp X-rays and have been genetically and molecularly characterized. In contrast to 3 H β-radiation, 100-kVp X-rays induced a bimodal distribution of Adh null mutations, intragenic mutations, ≤250 bp, and large deletions, >100 kb. A statistically significant difference was observed between the frequency of large deletions (23/23 or 1.0) induced by 3 H β-radiation and the frequency of large deletions (19/27 or 0.7) induced by 100-kVp X-rays. However, a statistical difference was not observed between the size distribution of the large deletions induced by 3 H β-radiation and X-rays. The relative deletion frequency (RDF) induced by 3 H β-radiation and 100-kVp X-rays was (1.0/0.7=1.4). The relative biological effectiveness (RBE) of these two radiation sources was 1.4, determined from the ratio of the regression coefficients of the respective 3 H β-radiation and X-ray sex-linked recessive lethal (SLRL) dose-response data. The large difference in size between the two classes of X-ray-induced Adh null mutations and the increase in mutation frequency and deletion frequency for 3 H β-radiation with respect to X-rays may indicate that the relative deletion frequency (RDF) is the molecular biological basis for the increase in the RBE for radiation sources with a mean LET value ≤10 keV/μm

  14. BCL2-like 11 intron 2 deletion polymorphism is not associated with non-small cell lung cancer risk and prognosis.

    Science.gov (United States)

    Cho, Eun Na; Kim, Eun Young; Jung, Ji Ye; Kim, Arum; Oh, In Jae; Kim, Young Chul; Chang, Yoon Soo

    2015-10-01

    BCL2-Like 11(BIM), which encodes a BH3-only protein, is a major pro-apoptotic molecule that facilitates cell death. We hypothesized that a BIM intron 2 deletion polymorphism increases lung cancer risk and predicts poor prognosis in non-small lung cancer (NSCLC) patients. We prospectively recruited 450 lung cancer patients and 1:1 age, sex, and smoking status matched control subjects from February 2013 to April 2014 among patients treated at Severance, Gangnam Severance, and Chonnam Hwasoon Hospital. The presence of a 2903-bp genomic DNA deletion polymorphism of intron 2 of BIM was analyzed by PCR and validated by sequencing. Odds ratios were calculated by chi-square tests and survival analysis with Kaplan-Meier estimation. Sixty-nine out of 450 (15.3%) lung cancer patients carried the BIM deletion polymorphism, while 66 out of 450 (14.7%) control subjects carried the BIM deletion polymorphism, with an odds ratio of for lung cancer of 1.054 (95% CI; 0.731-1.519). We categorized 406 NSCLC patients according to the presence of the polymorphism and found that there were no statistically significant differences in age, sex, histologic type, or stage between subjects with and without the deletion polymorphism. The BIM deletion polymorphism did not influence overall survival (OS) or progression free survival (PFS) in our sample (OS; 37.6 vs 34.4 months (P=0.759), PFS; 49.6 vs 26.0 months (P=0.434)). These findings indicate that the BIM deletion polymorphism is common in Korean NSCLC patients but does not significantly affect the intrinsic biologic function of BH3-only protein. Furthermore, the BIM deletion polymorphism did not predict clinical outcomes in patients with NSCLC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Physical mapping of a commonly deleted region, the site of a candidate tumor suppressor gene, at 12q22 in human male germ cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Murty, V.V.V.S.; Bosl, G.J.; Chaganti, R.S.K. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States)] [and others

    1996-08-01

    A candidate tumor suppressor gene (TSG) site at 12q22 characterized by a high frequency of loss of heterozygosity (LOH) and a homozygous deletion has previously (LOH) and a homozygous deletion has previously been reported in human male germ cell tumors (GCTs). In a detailed deletion mapping analysis of 67 normal-tumor DNAs utilizing 20 polymorphic markers mapped to 12q22-q24, we identified the limits of the minimal region of deletion at 12q22 between D12S377 (priximal) and D12S296 (distal). We have constructed a YAC contig map of a 3-cM region of this band between the proximal marker D12S101 and the distal marker D12S346, which contained the minimal region of deletion in GCTs. The map is composed of 53 overlapping YACs and 3 cosmids onto which 25 polymorphic and nonpolymorphic sequence-tagged sites (STSs) were placed in a unique order. The size of the minimal region of deletion was approximately 2 Mb from overlapping, nonchimeric YACs that spanned the region. We also developed a radiation hybrid (RH) map of the region between D12S101 and D12S346 containing 17 loci. The consensus order developed by RH mapping is in good agreement with the YAC STS-content map order. The RH map estimated the distance between D12S101 and D12S346 to be 246 cR{sub 8000} and the minimal region of deletion to be 141 cR{sub 8000}. In addition, four genes that were previously mapped to 12q22 have been excluded as candidate genes. The leads gained from the deletion mapping and physical maps should expedite the isolation and characterization of the TSG at 12q22. 35 refs., 4 figs., 2 tabs.

  16. Validation of the Internet Gaming Disorder Scale - Short-Form (IGDS9-SF) in an Italian-speaking sample.

    Science.gov (United States)

    Monacis, Lucia; Palo, Valeria de; Griffiths, Mark D; Sinatra, Maria

    2016-12-01

    Background and aims The inclusion of Internet Gaming Disorder (IGD) in Section III of the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders has increased the interest of researchers in the development of new standardized psychometric tools for the assessment of such a disorder. To date, the nine-item Internet Gaming Disorder Scale - Short-Form (IGDS9-SF) has only been validated in English, Portuguese, and Slovenian languages. Therefore, the aim of this investigation was to examine the psychometric properties of the IGDS9-SF in an Italian-speaking sample. Methods A total of 757 participants were recruited to the present study. Confirmatory factor analysis and multi-group analyses were applied to assess the construct validity. Reliability analyses comprised the average variance extracted, the standard error of measurement, and the factor determinacy coefficient. Convergent and criterion validities were established through the associations with other related constructs. The receiver operating characteristic curve analysis was used to determine an empirical cut-off point. Results Findings confirmed the single-factor structure of the instrument, its measurement invariance at the configural level, and the convergent and criterion validities. Satisfactory levels of reliability and a cut-off point of 21 were obtained. Discussion and conclusions The present study provides validity evidence for the use of the Italian version of the IGDS9-SF and may foster research into gaming addiction in the Italian context.

  17. Validation of the Internet Gaming Disorder Scale – Short-Form (IGDS9-SF) in an Italian-speaking sample

    Science.gov (United States)

    Monacis, Lucia; de Palo, Valeria; Griffiths, Mark D.; Sinatra, Maria

    2016-01-01

    Background and aims The inclusion of Internet Gaming Disorder (IGD) in Section III of the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders has increased the interest of researchers in the development of new standardized psychometric tools for the assessment of such a disorder. To date, the nine-item Internet Gaming Disorder Scale – Short-Form (IGDS9-SF) has only been validated in English, Portuguese, and Slovenian languages. Therefore, the aim of this investigation was to examine the psychometric properties of the IGDS9-SF in an Italian-speaking sample. Methods A total of 757 participants were recruited to the present study. Confirmatory factor analysis and multi-group analyses were applied to assess the construct validity. Reliability analyses comprised the average variance extracted, the standard error of measurement, and the factor determinacy coefficient. Convergent and criterion validities were established through the associations with other related constructs. The receiver operating characteristic curve analysis was used to determine an empirical cut-off point. Results Findings confirmed the single-factor structure of the instrument, its measurement invariance at the configural level, and the convergent and criterion validities. Satisfactory levels of reliability and a cut-off point of 21 were obtained. Discussion and conclusions The present study provides validity evidence for the use of the Italian version of the IGDS9-SF and may foster research into gaming addiction in the Italian context. PMID:27876422

  18. Selective deletion of cochlear hair cells causes rapid age-dependent changes in spiral ganglion and cochlear nucleus neurons.

    Science.gov (United States)

    Tong, Ling; Strong, Melissa K; Kaur, Tejbeer; Juiz, Jose M; Oesterle, Elizabeth C; Hume, Clifford; Warchol, Mark E; Palmiter, Richard D; Rubel, Edwin W

    2015-05-20

    During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival. Copyright © 2015 the authors 0270-6474/15/357878-14$15.00/0.

  19. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    International Nuclear Information System (INIS)

    Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

    2011-01-01

    Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

  20. Limited impact on glucose homeostasis of leptin receptor deletion from insulin- or proglucagon-expressing cells

    Directory of Open Access Journals (Sweden)

    Helen Soedling

    2015-09-01

    Conclusions/interpretation: The use here of a highly selective Cre recombinase indicates that leptin signalling plays a relatively minor, age- and sex-dependent role in the control of β cell function in the mouse. No in vivo role for leptin receptors on α cells, nor in other proglucagon-expressing cells, was detected in this study.

  1. Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.

    Directory of Open Access Journals (Sweden)

    Artur Brandt

    Full Text Available We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT in conjunction with a novel thermolabile mutant (U19dl89-97tsA58 of SV40 large T antigen (LT. This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.

  2. Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15

    Science.gov (United States)

    Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S.; Royer-Pokora, Brigitte

    2016-01-01

    We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells. PMID:27213811

  3. Genetic deletion of Cxcl14 in mice alters uterine NK cells

    International Nuclear Information System (INIS)

    Cao, Qichen; Chen, Hua; Deng, Zhili; Yue, Jingwen; Chen, Qi; Cao, Yujing; Ning, Lina; Lei, Xiaohua; Duan, Enkui

    2013-01-01

    Highlights: •We first examined the expression of Cxcl14 in MLAp and DB of uterus. •We found the uNK cells in MLAp and decidua express Cxcl14. •In Cxcl14 −/− placenta, we found significantly decreased uNK cells. •We first performed microarray to compare the gene expression in MLAp and DB. -- Abstract: The uterine natural killer cells (uNK cells) are the major immune cells in pregnant uterus and the number of uNK cells is dramatically increased during placentation and embryo development. The uNK cells are necessary for the immune tolerance, cytokine secretion and angiogenesis of placenta. Former studies indicated that the population expansion of uNK cells was accomplished through recruitment of NK cell precursors from the spleen and bone marrow, but not proliferation of NK cells. However, the necessary molecules within this process were little understood. Here in our study, we found the co-localized expression of Cxcl14 protein with uNK cells in E13.5 pregnant uterus. Moreover, we used Cxcl14 knockout mice to examine uNK cells in mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) of E13.5 pregnant uterus and found significantly decreased uNK cells in Cxcl14 −/− pregnant uteri compared with Cxcl14 +/− pregnant uteri. To further explorer the molecular change in MLAp and DB after Cxcl14 knockout, we isolated the MLAp and DB from Cxcl14 +/+ and Cxcl14 −/− pregnant uteri and performed microarray analysis. We found many genes were up and down regulated after Cxcl14 knockout. In conclusion, our results suggested the important function of Cxcl14 in uNK cells and the proper level of Cxcl14 protein were required to recruit NK cells to pregnant uterus

  4. Genetic deletion of Cxcl14 in mice alters uterine NK cells

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Qichen [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan, Beijing 100049 (China); Chen, Hua [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101 (China); Deng, Zhili [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan, Beijing 100049 (China); Yue, Jingwen; Chen, Qi; Cao, Yujing; Ning, Lina; Lei, Xiaohua [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101 (China); Duan, Enkui, E-mail: duane@ioz.ac.cn [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101 (China)

    2013-06-14

    Highlights: •We first examined the expression of Cxcl14 in MLAp and DB of uterus. •We found the uNK cells in MLAp and decidua express Cxcl14. •In Cxcl14{sup −/−} placenta, we found significantly decreased uNK cells. •We first performed microarray to compare the gene expression in MLAp and DB. -- Abstract: The uterine natural killer cells (uNK cells) are the major immune cells in pregnant uterus and the number of uNK cells is dramatically increased during placentation and embryo development. The uNK cells are necessary for the immune tolerance, cytokine secretion and angiogenesis of placenta. Former studies indicated that the population expansion of uNK cells was accomplished through recruitment of NK cell precursors from the spleen and bone marrow, but not proliferation of NK cells. However, the necessary molecules within this process were little understood. Here in our study, we found the co-localized expression of Cxcl14 protein with uNK cells in E13.5 pregnant uterus. Moreover, we used Cxcl14 knockout mice to examine uNK cells in mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) of E13.5 pregnant uterus and found significantly decreased uNK cells in Cxcl14{sup −/−} pregnant uteri compared with Cxcl14{sup +/−} pregnant uteri. To further explorer the molecular change in MLAp and DB after Cxcl14 knockout, we isolated the MLAp and DB from Cxcl14{sup +/+} and Cxcl14{sup −/−} pregnant uteri and performed microarray analysis. We found many genes were up and down regulated after Cxcl14 knockout. In conclusion, our results suggested the important function of Cxcl14 in uNK cells and the proper level of Cxcl14 protein were required to recruit NK cells to pregnant uterus.

  5. Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae.

    Science.gov (United States)

    Kobayashi, Takuji; Maeda, Hiroshi; Takeuchi, Michio; Yamagata, Youhei

    2017-05-01

    Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ∆admA∆admB and ∆admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ∆admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ∆admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.

  6. The conceptualisation and measurement of DSM-5 Internet Gaming Disorder: the development of the IGD-20 Test.

    Directory of Open Access Journals (Sweden)

    Halley M Pontes

    Full Text Available Over the last decade, there has been growing concern about 'gaming addiction' and its widely documented detrimental impacts on a minority of individuals that play excessively. The latest (fifth edition of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-5 included nine criteria for the potential diagnosis of Internet Gaming Disorder (IGD and noted that it was a condition that warranted further empirical study.The main aim of this study was to develop a valid and reliable standardised psychometrically robust tool in addition to providing empirically supported cut-off points.A sample of 1003 gamers (85.2% males; mean age 26 years from 57 different countries were recruited via online gaming forums. Validity was assessed by confirmatory factor analysis (CFA, criterion-related validity, and concurrent validity. Latent profile analysis was also carried to distinguish disordered gamers from non-disordered gamers. Sensitivity and specificity analyses were performed to determine an empirical cut-off for the test.The CFA confirmed the viability of IGD-20 Test with a six-factor structure (salience, mood modification, tolerance, withdrawal, conflict and relapse for the assessment of IGD according to the nine criteria from DSM-5. The IGD-20 Test proved to be valid and reliable. According to the latent profile analysis, 5.3% of the total participants were classed as disordered gamers. Additionally, an optimal empirical cut-off of 71 points (out of 100 seemed to be adequate according to the sensitivity and specificity analyses carried.The present findings support the viability of the IGD-20 Test as an adequate standardised psychometrically robust tool for assessing internet gaming disorder. Consequently, the new instrument represents the first step towards unification and consensus in the field of gaming studies.

  7. The Conceptualisation and Measurement of DSM-5 Internet Gaming Disorder: The Development of the IGD-20 Test

    Science.gov (United States)

    Pontes, Halley M.; Király, Orsolya; Demetrovics, Zsolt; Griffiths, Mark D.

    2014-01-01

    Background Over the last decade, there has been growing concern about ‘gaming addiction’ and its widely documented detrimental impacts on a minority of individuals that play excessively. The latest (fifth) edition of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-5) included nine criteria for the potential diagnosis of Internet Gaming Disorder (IGD) and noted that it was a condition that warranted further empirical study. Aim: The main aim of this study was to develop a valid and reliable standardised psychometrically robust tool in addition to providing empirically supported cut-off points. Methods A sample of 1003 gamers (85.2% males; mean age 26 years) from 57 different countries were recruited via online gaming forums. Validity was assessed by confirmatory factor analysis (CFA), criterion-related validity, and concurrent validity. Latent profile analysis was also carried to distinguish disordered gamers from non-disordered gamers. Sensitivity and specificity analyses were performed to determine an empirical cut-off for the test. Results The CFA confirmed the viability of IGD-20 Test with a six-factor structure (salience, mood modification, tolerance, withdrawal, conflict and relapse) for the assessment of IGD according to the nine criteria from DSM-5. The IGD-20 Test proved to be valid and reliable. According to the latent profile analysis, 5.3% of the total participants were classed as disordered gamers. Additionally, an optimal empirical cut-off of 71 points (out of 100) seemed to be adequate according to the sensitivity and specificity analyses carried. Conclusions The present findings support the viability of the IGD-20 Test as an adequate standardised psychometrically robust tool for assessing internet gaming disorder. Consequently, the new instrument represents the first step towards unification and consensus in the field of gaming studies. PMID:25313515

  8. The conceptualisation and measurement of DSM-5 Internet Gaming Disorder: the development of the IGD-20 Test.

    Science.gov (United States)

    Pontes, Halley M; Király, Orsolya; Demetrovics, Zsolt; Griffiths, Mark D

    2014-01-01

    Over the last decade, there has been growing concern about 'gaming addiction' and its widely documented detrimental impacts on a minority of individuals that play excessively. The latest (fifth) edition of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-5) included nine criteria for the potential diagnosis of Internet Gaming Disorder (IGD) and noted that it was a condition that warranted further empirical study. The main aim of this study was to develop a valid and reliable standardised psychometrically robust tool in addition to providing empirically supported cut-off points. A sample of 1003 gamers (85.2% males; mean age 26 years) from 57 different countries were recruited via online gaming forums. Validity was assessed by confirmatory factor analysis (CFA), criterion-related validity, and concurrent validity. Latent profile analysis was also carried to distinguish disordered gamers from non-disordered gamers. Sensitivity and specificity analyses were performed to determine an empirical cut-off for the test. The CFA confirmed the viability of IGD-20 Test with a six-factor structure (salience, mood modification, tolerance, withdrawal, conflict and relapse) for the assessment of IGD according to the nine criteria from DSM-5. The IGD-20 Test proved to be valid and reliable. According to the latent profile analysis, 5.3% of the total participants were classed as disordered gamers. Additionally, an optimal empirical cut-off of 71 points (out of 100) seemed to be adequate according to the sensitivity and specificity analyses carried. The present findings support the viability of the IGD-20 Test as an adequate standardised psychometrically robust tool for assessing internet gaming disorder. Consequently, the new instrument represents the first step towards unification and consensus in the field of gaming studies.

  9. Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2015-12-01

    performed the clonogenic survival assay using LAPC4 as a model as these cell form well defined colonies compared to C42B cells. This assay revealed that...cells. Future studies with other PTEN deficient models are important to validate the effectiveness of WEE1 inhibitors to tackle recurrent PC...Amplifications and missense mutations Cixutumumab (IMC-A12), Dalotuzumab (MK-0646; h7C10), Linsitinib ( OSI -906) EPHA5 12% LAC, 10% SCLC, 9% SAC, Lung

  10. Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Magdalena Radwanska

    2007-05-01

    Full Text Available Effector responses induced by polarized CD4+ T helper 2 (Th2 cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha. IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(creIL-4Ralpha(-/lox deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(creIL-4Ralpha(-/lox mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(creIL-4Ralpha(-/lox mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L

  11. Deletion of Foxp3+ regulatory T cells in genetically targeted mice supports development of intestinal inflammation

    Directory of Open Access Journals (Sweden)

    Boehm Franziska

    2012-07-01

    Full Text Available Abstract Background Mice lacking Foxp3+ regulatory T (Treg cells develop severe tissue inflammation in lung, skin, and liver with premature death, whereas the intestine remains uninflamed. This study aims to demonstrate the importance of Foxp3+ Treg for the activation of T cells and the development of intestinal inflammation. Methods Foxp3-GFP-DTR (human diphtheria toxin receptor C57BL/6 mice allow elimination of Foxp3+ Treg by treatment with Dx (diphtheria toxin. The influence of Foxp3+ Treg on intestinal inflammation was tested using the CD4+ T-cell transfer colitis model in Rag−/− C57BL/6 mice and the acute DSS-colitis model. Results Continuous depletion of Foxp3+ Treg in Foxp3-GFP-DTR mice led to dramatic weight loss and death of mice by day 28. After 10 days of depletion of Foxp3+ Treg, isolated CD4+ T-cells were activated and produced extensive amounts of IFN-γ, IL-13, and IL-17A. Transfer of total CD4+ T-cells isolated from Foxp3-GFP-DTR mice did not result in any changes of intestinal homeostasis in Rag−/− C57BL/6 mice. However, administration of DTx between days 14 and 18 after T-cell reconstitution, lead to elimination of Foxp3+ Treg and to immediate weight loss due to intestinal inflammation. This pro-inflammatory effect of Foxp3+ Treg depletion consecutively increased inflammatory cytokine production. Further, the depletion of Foxp3+ Treg from Foxp3-GFP-DTR mice increased the severity of acute dSS-colitis accompanied by 80% lethality of Treg-depleted mice. CD4+ effector T-cells from Foxp3+ Treg-depleted mice produced significantly more pro-inflammatory cytokines. Conclusion Intermittent depletion of Foxp3+ Treg aggravates intestinal inflammatory responses demonstrating the importance of Foxp3+ Treg for the balance at the mucosal surface of the intestine.

  12. Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

    Science.gov (United States)

    Yap, Jin Yan; Wirasinha, Rushika C; Chan, Anna; Howard, Debbie R; Goodnow, Christopher C; Daley, Stephen R

    2018-02-07

    Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 + regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes. © 2018 John Wiley & Sons Ltd.

  13. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  14. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike

    2006-01-01

    Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines ...

  15. A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

    Science.gov (United States)

    Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

    2018-04-01

    The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

  16. Enteric nervous system specific deletion of Foxd3 disrupts glial cell differentiation and activates compensatory enteric progenitors.

    Science.gov (United States)

    Mundell, Nathan A; Plank, Jennifer L; LeGrone, Alison W; Frist, Audrey Y; Zhu, Lei; Shin, Myung K; Southard-Smith, E Michelle; Labosky, Patricia A

    2012-03-15

    The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial

  17. Analysis of mtDNT 4977bp deletion induced by ionizing radiation in human peripheral blood nucleated cells using real-time PCR

    International Nuclear Information System (INIS)

    Fan Tianli; Wang Ping; Han Lin; Liu Yulong; Liu Yumin

    2010-01-01

    To detect mitochondrial DNA(mtDNA) 4977bp deletion(triangle open mtDNA 4977 ) in human peripheral blood nucleated cells exposed to ionizing radiation in vitro by using real-time PCR, and explore possibility of the index as biodosimetry for estimating biological dose in radiation accident,six healthy individuals' peripheral blood was collected,and the blood samples were irradiated with 0,1,2,3,4 and 5 Gy 60 Co gamma-ray. The triangle open mtDNA 4977 and total mtDNA copy number(mtDNA total ) in the mtDNA samples were detected, and then the deletion rates were calculated. The results showed that the mtDNA total and triangle open mtDNA 4977 copy number, and the deletion rates of mtDNA 4977bp in the mtDNA samples from 6 healthy individuals' blood exposed to 1-5 Gy radiation were higher than that with the samples exposed to 0 Gy radiation(p 0.05). The results indicated that ionizing radiation can induce accumulation of the triangle open mtDNA 4977 and increase of mtDNA total copy number in human peripheral blood nucleated cells,but both the mtDNA 4977bp deletion and exposure dose(0-5 Gy) were not obviously correlated. (authors)

  18. Tolerance without clonal expansion: self-antigen-expressing B cells program self-reactive T cells for future deletion.

    Science.gov (United States)

    Frommer, Friederike; Heinen, Tobias J A J; Wunderlich, F Thomas; Yogev, Nir; Buch, Thorsten; Roers, Axel; Bettelli, Estelle; Müller, Werner; Anderton, Stephen M; Waisman, Ari

    2008-10-15

    B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells.

  19. IGDS/TRAP Interface Program (ITIP). Detailed Design Specification (DDS). [network flow diagrams for coal gasification studies

    Science.gov (United States)

    Jefferys, S.; Johnson, W.; Lewis, R.; Rich, R.

    1981-01-01

    The software modules which comprise the IGDS/TRAP Interface Program are described. A hierarchical input processing output (HIPO) chart for each user command is given. The description consists of: (1) function of the user command; (2) calling sequence; (3) moduls which call this use command; (4) modules called by this user command; (5) IGDS commands used by this user command; and (6) local usage of global registers. Each HIPO contains the principal functions performed within the module. Also included with each function are a list of the inputs which may be required to perform the function and a list of the outputs which may be created as a result of performing the function.

  20. The effect of conditional EFNB1 deletion in the T cell compartment on T cell development and function

    Directory of Open Access Journals (Sweden)

    Jin Wei

    2011-12-01

    Full Text Available Abstract Background Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs, are also cell surface molecules. Ephs interact with EFNs transmitting signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. EFNB1 is known to be able to co-stimulate T cells in vitro and to modulate thymocyte development in a model of foetal thymus organ culture. To further understand the role of EFNB1 in T cell immunity, we generated T-cell-specific EFNB1 gene knockout mice to assess T cell development and function in these mice. Results The mice were of normal size and cellularity in the thymus and spleen and had normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT control mice repopulated host spleen T cell pool to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells showed an ability to differentiate into Th1, Th2, Th17 and Treg cells similar to control CD4 cells. Conclusions Our results suggest that the function of EFNB1 in the T cell compartment could be compensated by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 in T cell development and function.

  1. The Conceptualisation and Measurement of DSM-5 Internet Gaming Disorder: The Development of the IGD-20 Test

    OpenAIRE

    Pontes, Halley M.; Király, Orsolya; Demetrovics, Zsolt; Griffiths, Mark D.

    2014-01-01

    Background Over the last decade, there has been growing concern about ‘gaming addiction’ and its widely documented detrimental impacts on a minority of individuals that play excessively. The latest (fifth) edition of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-5) included nine criteria for the potential diagnosis of Internet Gaming Disorder (IGD) and noted that it was a condition that warranted further empirical study. Aim: The main aim of...

  2. „NS-Raubgut aus zweiter Hand“ - Provenienzrecherchen in der Bibliothek des IGdJ

    Directory of Open Access Journals (Sweden)

    Jörn Kreuzer

    2014-12-01

    Full Text Available Das Institut für die Geschichte der deutschen Juden in Hamburg führt im Rahmen des Projekts "NS-Raubgut in der Bibliothek des IGdJ" umfangreiche Recherchen in seinen Buchbeständen durch. Die Untersuchung fügt sich in die Reihe von Forschungsvorhaben, die in der Folge der "Washingtoner Konferenz über Vermögenswerte aus der Zeit des Holocaust" (1998 und der ein Jahr später verabschiedeten "Erklärung der Bundesregierung, der Länder und der kommunalen Spitzenverbände zur Auffindung und zur Rückgabe NS-verfolgungsbedingt entzogenen Kulturgutes, insbesondere aus jüdischem Besitz" in diversen deutschen Bibliotheken, Museen, Archiven und anderen kulturellen Einrichtungen durchgeführt werden. Zurzeit befinden sich in der Bibliothek des IGdJ rund 50.000 Bände. Im Grunde ist bei allen Werken, die vor 1945 erschienen sind, eine Provenienz aus NS-Raub- bzw. NS-Beutegutbeständen möglich. Eine systematische Erfassung und Bearbeitung dieses rund 6.000 bis 9.000 Bände umfassenden Bestandes ist bislang nicht erfolgt. Da die Institutsbibliothek als jüdische Sammlung konzipiert und aufgebaut wurde, verstärkt sich diese Vermutung. Weil das Institut erst 1966 gegründet wurde, handelt es sich wahrscheinlich um "NS-Raubgut aus zweiter Hand“. Eine weitere Aufgabenstellung ergibt sich aus der Tatsache, dass in den Anfangsjahren der Bibliothek keine Zugangsjournale geführt wurden. Somit können in vielen Fällen nur die Bücher selbst Hinweise auf ihre Herkunft geben, weshalb als erste Maßnahme die eingehende Buchautopsie anhand des Zettelkatalogs durchgeführt wird. Der systematischen Suche nach NS-verfolgungsbedingt entzogenem Kulturgut folgt die Dokumentation und Bekanntgabe der Ergebnisse mit dem Ziel der Restitution an die Vorbesitzer oder deren Erben. In einem Werkstattbericht werden erste Ergebnisse vorgestellt. The Institute of the History of the German Jews (Institut für die Geschichte der deutschenJuden, IGdJ is currently conducting a

  3. Deletion of Menin in craniofacial osteogenic cells in mice elicits development of mandibular ossifying fibroma.

    Science.gov (United States)

    Lee, S; Liu, P; Teinturier, R; Jakob, J; Tschaffon, M; Tasdogan, A; Wittig, R; Hoeller, S; Baumhoer, D; Frappart, L; Vettorazzi, S; Bertolino, P; Zhang, C; Tuckermann, J

    2018-02-01

    Ossifying fibroma (OF) is a rare benign tumor of the craniofacial bones that can reach considerable and disfiguring dimensions if left untreated. Although the clinicopathological characteristics of OF are well established, the underlying etiology has remained largely unknown. Our work indicates that Men1-a tumor suppressor gene responsible of Multiple endocrine neoplasia type 1-is critical for OF formation and shows that mice with targeted disruption of Men1 in osteoblasts (Men1 Runx2Cre ) develop multifocal OF in the mandible with a 100% penetrance. Using lineage-tracing analysis, we demonstrate that loss of Men1 arrests stromal osteoprogenitors in OF at the osterix-positive pre-osteoblastic differentiation stage. Analysis of Men1-lacking stromal spindle cells isolated from OF (OF-derived MSCs (OFMSCs)) revealed a downregulation of the cyclin-dependent kinase (CDK) inhibitor Cdkn1a, consistent with an increased proliferation rate. Intriguingly, the re-expression of Men1 in Men1-deficient OFMSCs restored Cdkn1a expression and abrogated cellular proliferation supporting the tumor-suppressive role of Men1 in OF. Although our work presents the first evidence of Men1 in OF development, it further provides the first genetic mouse model of OF that can be used to better understand the molecular pathogenesis of these benign tumors and to potentially develop novel treatment strategies.

  4. Measurement Invariance of the Internet Gaming Disorder Scale-Short-Form (IGDS9-SF) between the United States of America, India and the United Kingdom.

    Science.gov (United States)

    Pontes, Halley M; Stavropoulos, Vasileios; Griffiths, Mark D

    2017-11-01

    The Internet Gaming Disorder Scale-Short-Form (IGDS9-SF) has been extensively used worldwide to assess Internet Gaming Disorder (IGD) behaviors. Therefore, investigating cultural limitations and implications in its applicability is necessary. The cross-cultural feasibility of a test can be psychometrically evaluated with measurement invariance analyses. Thus, the present study used Multigroup Confirmatory Factor Analysis (MGCFA) to examine the IGDS9-SF measurement invariance across gamers from the United States of America (USA), India, and the United Kingdom (UK). A total of 1013 gamers from the USA (n = 405), India (n = 336), and the UK (n = 272) were recruited. Although the one-factor structure of the IGD construct was supported, cross-country variations were demonstrated considering the way that this was reflected on items assessing preoccupation/salience, tolerance, deception, gaming escapism/mood modification, as well as daily activities' impairment related to gaming. Furthermore, the same scores on items assessing withdrawal symptoms, tolerance, lack of control over gaming engagement, escapism/mood modification and daily activities impairment associated to gaming, have been found to reflect various levels of IGD severity across the three groups. The implications of these results are further discussed in the context of existing evidence regarding the assessment of IGD. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Dendritic Cell-Specific Deletion of β-Catenin Results in Fewer Regulatory T-Cells without Exacerbating Autoimmune Collagen-Induced Arthritis.

    Science.gov (United States)

    Alves, C Henrique; Ober-Blöbaum, Julia L; Brouwers-Haspels, Inge; Asmawidjaja, Patrick S; Mus, Adriana M C; Razawy, Wida; Molendijk, Marlieke; Clausen, Björn E; Lubberts, Erik

    2015-01-01

    Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

  6. Dendritic Cell-Specific Deletion of β-Catenin Results in Fewer Regulatory T-Cells without Exacerbating Autoimmune Collagen-Induced Arthritis.

    Directory of Open Access Journals (Sweden)

    C Henrique Alves

    Full Text Available Dendritic cells (DCs are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA. Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs (CD4+CD25highFoxP3+, but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+, Th2 (CCR6-CXCR3-CCR4+ and Th1 (CCR6-CXCR3+CCR4- cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

  7. Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD.

    Science.gov (United States)

    Zhang, Le; Willemse, Joost; Hoskisson, Paul A; van Wezel, Gilles P

    2018-05-09

    Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

  8. Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters

    Science.gov (United States)

    Borghei, Yasaman-Sadat; Hosseini, Morteza; Ganjali, Mohammad Reza

    2018-01-01

    Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10-10 to 2.4 × 10-6 M with a detection limit (LOD) of 6.4 × 10-11 M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

  9. [Polymorphisms of KITLG, SPRY4, and BAK1 genes in patients with testicular germ cell tumors and individuals with infertility associated with AZFc deletion of the Y chromosome].

    Science.gov (United States)

    Nemtsova, M V; Ivkin, E V; Simonova, O A; Rudenko, V V; Chernykh, V B; Mikhaylenko, D S; Loran, O B

    2016-01-01

    Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44-19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11-12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to

  10. Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

    DEFF Research Database (Denmark)

    Li, Xiyang; Guo, Li; Deng, Ling

    2011-01-01

    Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly...... results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell....

  11. Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines.

    Science.gov (United States)

    Klorin, Geula; Rozenblum, Ester; Glebov, Oleg; Walker, Robert L; Park, Yoonsoo; Meltzer, Paul S; Kirsch, Ilan R; Kaye, Frederic J; Roschke, Anna V

    2013-05-01

    High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired. Published by Elsevier Inc.

  12. DNA Fragmentation Factor 45 (DFF45 Gene at 1p36.2 Is Homozygously Deleted and Encodes Variant Transcripts in Neuroblastoma Cell Line

    Directory of Open Access Journals (Sweden)

    Hong Wei Yang

    2001-01-01

    Full Text Available Recently, loss of heterozygosity (LOH studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p in neuroblastoma (NB. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1 p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45 gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT-polymerase chain reaction (PCR and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

  13. T-cell receptor and K-deleting recombination excision circles in newborn screening of T- and B-cell defects: review of the literature and future challenges

    Directory of Open Access Journals (Sweden)

    Marco Chiarini

    2013-05-01

    Full Text Available Since its introduction as a public health programme in the United States in the early 1960s, newborn blood screening (NBS has evolved from the detection of phenylalanine levels on filter paper to the application of DNA-based technologies to identify T-cell lymphopenia in infants with severe combined immunodeficiency. This latter use of NBS has required the development of an assay for T-cell lymphopenia based on the quantification of T-cell receptor excision circles (TRECs that could be performed on dried blood spots routinely collected from newborn infants. The TREC-based NBS was developed six years ago, and there have already been 7 successful pilot studies since then. Similarly, efforts are now being made to establish a screen for B-cell defects, in particular agammaglobulinaemia, taking advantage of the introduction of the method for the quantification of K-deleting recombination excision circles (KRECs. A further achievement of NBS could be the simultaneous recognition of T- and B-cell defects using the combined quantification of TRECs and KRECs from Guthrie card blood spots. This approach may help the early identification of infants with T- and B-cell deficiencies so that they can then be referred to specialised paediatric centres, where a precise diagnosis of severe combined immunodeficiency and agammaglobulinaemia can be performed, and where then they can immediately receive specific therapy. Simultaneous TREC and KREC quantification should also allow classification of patients into subgroups and help identify children with less serious primary immunodeficiencies. This would help avoid the opportunistic infections and frequent hospitalisations that result from a late or lack of diagnosis.

  14. High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Sven Reiche

    Full Text Available The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs originated from 26 and the kappa light chains (LCs from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4% in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

  15. The E7-associated cell-surface antigen: a marker for the 11p13 chromosomal deletion associated with aniridia-Wilms tumor.

    OpenAIRE

    Scoggin, C H; Fisher, J H; Shoemaker, S A; Morse, H; Leigh, T; Riccardi, V M

    1985-01-01

    Unbalanced interstitial deletions of the p13 region of human chromosome 11 have been associated with congenital hypoplasia or aplasia of the iris, mental retardation, ambiguous genitalia, and predisposition to Wilms tumor of the kidney. Utilizing somatic cell hybrids containing either the normal or abnormal chromosome 11 from a child with Wilms tumor and aniridia, we previously mapped the E7 cell-surface antigen to the 11p1300-to-11p15.1 region. To localize even further the site of this antig...

  16. Hilar granule cells of the mouse dentate gyrus: effects of age, septotemporal location, strain, and selective deletion of the proapoptotic gene BAX.

    Science.gov (United States)

    Bermudez-Hernandez, Keria; Lu, Yi-Ling; Moretto, Jillian; Jain, Swati; LaFrancois, John J; Duffy, Aine M; Scharfman, Helen E

    2017-09-01

    The dentate gyrus (DG) principal cells are glutamatergic granule cells (GCs), and they are located in a compact cell layer. However, GCs are also present in the adjacent hilar region, but have been described in only a few studies. Therefore, we used the transcription factor prospero homeobox 1 (Prox1) to quantify GCs at postnatal day (PND) 16, 30, and 60 in a common mouse strain, C57BL/6J mice. At PND16, there was a large population of Prox1-immunoreactive (ir) hilar cells, with more in the septal than temporal hippocampus. At PND30 and 60, the size of the hilar Prox1-ir cell population was reduced. Similar numbers of hilar Prox1-expressing cells were observed in PND30 and 60 Swiss Webster mice. Prox1 is usually considered to be a marker of postmitotic GCs. However, many Prox1-ir hilar cells, especially at PND16, were not double-labeled with NeuN, a marker typically found in mature neurons. Most hilar Prox1-positive cells at PND16 co-expressed doublecortin (DCX) and calretinin, markers of immature GCs. Double-labeling with a marker of actively dividing cells, Ki67, was not detected. These results suggest that, surprisingly, a large population of cells in the hilus at PND16 are immature GCs (Type 2b and Type 3 cells). We also asked whether hilar Prox1-ir cell numbers are modifiable. To examine this issue, we conditionally deleted the proapoptotic gene BAX in Nestin-expressing cells at a time when there are numerous immature GCs in the hilus, PND2-8. When these mice were examined at PND60, the numbers of Prox1-ir hilar cells were significantly increased compared to control mice. However, deletion of BAX did not appear to change the proportion that co-expressed NeuN, suggesting that the size of the hilar Prox1-expressing population is modifiable. However, deleting BAX, a major developmental disruption, does not appear to change the proportion that ultimately becomes neurons.

  17. T-cell transfer and cytokine/TCR gene deletion models in the study of inflammatory bowel disease

    DEFF Research Database (Denmark)

    Bregenholt, S; Delbro, D; Claesson, Mogens Helweg

    1997-01-01

    Until recently there existed no appropriate immunological animal models for human inflammatory bowel diseases (IBD). Today a number of models, mostly in the mouse and rat, have proved useful in the study of several aspects of IBD, including the histopathology and the disease-inductive and -protec...... and in gene-deleted mice....

  18. Low thymic output in the 22q11.2 deletion syndrome measured by CCR9+CD45RA+ T cell counts and T cell receptor rearrangement excision circles

    DEFF Research Database (Denmark)

    Lima, K; Abrahamsen, Gitte Meldgaard; Foelling, I

    2010-01-01

    Thymic hypoplasia is a frequent feature of the 22q11.2 deletion syndrome, but we know little about patients' age-related thymic output and long-term consequences for their immune system. We measured the expression of T cell receptor rearrangement excision circles (TREC) and used flow cytometry...

  19. CRISPR-Mediated Genomic Deletion of Sox2 in the Axolotl Shows a Requirement in Spinal Cord Neural Stem Cell Amplification during Tail Regeneration

    Directory of Open Access Journals (Sweden)

    Ji-Feng Fei

    2014-09-01

    Full Text Available The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs and clustered regularly interspaced short palindromic repeats (CRISPRs in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.

  20. Functional clonal deletion versus suppressor cell-induced transplantation tolerance in chimeras prepared with a short course of total-lymphoid irradiation

    International Nuclear Information System (INIS)

    Slavin, S.; Morecki, S.; Weigensberg, M.; Bar, S.; Weiss, L.

    1986-01-01

    Allogeneic bone marrow (BM) chimeras induced by infusion of BM cells into recipients conditioned with total lymphoid irradiation (TLI) were shown to develop humoral and cell-mediated tolerance to host and donor-type alloantigens by a number of in vitro and in vivo assays. Spleen cells of tolerant chimeras exhibited suppressive activity of mixed lymphocyte reaction (MLR). MLR suppression was not abrogated by depletion of Lyt-2 cells, and neither could Lyt-2-positive cells sorted from the spleens of tolerant chimeras suppress MLR or attenuate graft-versus-host reactivity in vivo. Likewise, specifically unresponsive spleen cells obtained from chimeras could not be induced to respond in MLR against tolerizing host-type cells following depletion of Lyt-2 or passage through a nylon-wool column. Tolerance of chimera spleen cells to host alloantigens, best documented by permanent survival of donor-type skin allografts, could be adoptively transferred into syngeneic recipients treated by heavy irradiation but not into untreated or mildly irradiated recipients. Adoptive transfer of tolerance seemed to be associated with experimental conditions favoring engraftment of tolerant cells rather than suppression of host reactivity. We speculate that although host and/or donor-derived suppressor cells may be operating in reducing the pool of specific alloreactive clones by blocking cell proliferation in response to allogeneic challenge, the final outcome in tolerant chimeras is actual or functional deletion of alloreactive clones

  1. Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

    OpenAIRE

    Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

    2007-01-01

    Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as in...

  2. Deletion of lipoprotein PG0717 in Porphyromonas gingivalis W83 reduces gingipain activity and alters trafficking in and response by host cells.

    Directory of Open Access Journals (Sweden)

    Leticia Reyes

    Full Text Available P. gingivalis (Pg, a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC, suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp and lysine (Kgp gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.

  3. A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

    Science.gov (United States)

    Naveilhan, P; Baudet, C; Jabbour, W; Wion, D

    1994-09-01

    A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

  4. Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

    Science.gov (United States)

    Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

    2010-02-01

    The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

  5. Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming.

    Directory of Open Access Journals (Sweden)

    Quan Wu

    2016-09-01

    Full Text Available The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4 in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells.

  6. Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming

    Science.gov (United States)

    Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

    2016-01-01

    The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells. PMID:27606421

  7. A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

    Science.gov (United States)

    Sano, R; Kuboya, E; Nakajima, T; Takahashi, Y; Takahashi, K; Kubo, R; Kominato, Y; Takeshita, H; Yamao, H; Kishida, T; Isa, K; Ogasawara, K; Uchikawa, M

    2015-04-01

    We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently. © 2014 International Society of Blood Transfusion.

  8. AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

    Science.gov (United States)

    Virts, Elizabeth L; Jankowska, Anna; Mackay, Craig; Glaas, Marcel F; Wiek, Constanze; Kelich, Stephanie L; Lottmann, Nadine; Kennedy, Felicia M; Marchal, Christophe; Lehnert, Erik; Scharf, Rüdiger E; Dufour, Carlo; Lanciotti, Marina; Farruggia, Piero; Santoro, Alessandra; Savasan, Süreyya; Scheckenbach, Kathrin; Schipper, Jörg; Wagenmann, Martin; Lewis, Todd; Leffak, Michael; Farlow, Janice L; Foroud, Tatiana M; Honisch, Ellen; Niederacher, Dieter; Chakraborty, Sujata C; Vance, Gail H; Pruss, Dmitry; Timms, Kirsten M; Lanchbury, Jerry S; Alpi, Arno F; Hanenberg, Helmut

    2015-09-15

    Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene. © The Author 2015. Published by Oxford University Press.

  9. [MVK gene abnormality and new approach to treatment of hyper IgD syndrome and periodic fever syndrome].

    Science.gov (United States)

    Naruto, Takuya

    2007-04-01

    Hyper IgD and periodic fever syndrome (HIDS; OMIM 260920) is one of the hereditary autoinflammatory syndromes characterized by recurrent episodes of fever and inflammation.. HIDS is an autosomal recessive disorder characterized by recurrent fever attacks in early childhood. HIDS caused by mevalonate kinase (MK) mutations, also that is the gene of mevalonic aciduria (OMIM 251170). During febrile episodes, urinary mevalonate concentrations were found to be significantly elevated in patients. Diagnosis of HIDS was retrieving gene or measurement of the enzyme activity in peripheral blood lymphocyte in general. This of HIDS is an activity decline of MK, and a complete deficiency of MK becomes a mevalonic aciduria with a nervous symptom. The relation between the fever and inflammation of mevalonate or isoprenoid products are uncertain. The therapy attempt with statins, which is inhibited the next enzyme after HMG-CoA reductase, or inhibit the proinflammatory cytokines.

  10. Deletion of AU-rich elements within the Bcl2 3'UTR reduces protein expression and B cell survival in vivo.

    Directory of Open Access Journals (Sweden)

    Manuel D Díaz-Muñoz

    Full Text Available Post-transcriptional mRNA regulation by RNA binding proteins (RBPs associated with AU-rich elements (AREs present in the 3' untranslated region (3'UTR of specific mRNAs modulates transcript stability and translation in eukaryotic cells. Here we have functionally characterised the importance of the AREs present within the Bcl2 3'UTR in order to maintain Bcl2 expression. Gene targeting deletion of 300 nucleotides of the Bcl2 3'UTR rich in AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing cell lifespan. Generation of chimeric mice indicates that Bcl2-ARE∆/∆ B cells have an intrinsic competitive disadvantage compared to wild type cells. Biochemical assays and predictions using a bioinformatics approach show that several RBPs bind to the Bcl2 AREs, including AUF1 and HuR proteins. Altogether, association of RBPs to Bcl2 AREs contributes to Bcl2 protein expression by stabilizing Bcl2 mRNA and promotes B cell maintenance.

  11. Giant Cell Arteritis of the Female Genital Tract With Occult Temporal Arteritis and Marginal Zone Lymphoma Harboring Novel 20q Deletion: A Case Report and Literature Review.

    Science.gov (United States)

    Pradhan, Dinesh; Amin, Rajnikant M; Jones, Miroslawa W; Surti, Urvashi; Parwani, Anil V

    2016-02-01

    Giant cell arteritis (GCA) is an immunologically mediated vasculitis of large and medium-sized vessels, typically affecting the cranial arteries and usually occurring in the elderly. GCA of the female genital tract is extremely rare with only 31 cases reported in the English literature. An 83-year-old white female with postmenopausal vaginal bleeding revealed an endometrial polyp on pelvic ultrasonography following which polypectomy and subsequently hysterectomy with bilateral salpingo-oophorectomy was done. Microscopy revealed a well-differentiated endometrioid adenocarcinoma. Interestingly, classic GCA involving numerous small to medium-sized arteries of the cervix, myometrium, bilateral fallopian tubes, and ovaries was also identified. Hematologic evaluation revealed marginal zone lymphoma with an exceptionally rare 20q deletion. Bilateral temporal artery biopsy was done subsequently, which exhibited GCA on microscopy. Corticosteroid was started that improved her polymyalgia rheumatica symptoms. The patient is on follow-up for 3 years and is doing well. To our knowledge, this is the first case of GCA of the female genital tract associated with a lymphoma and the second case of marginal zone lymphoma with the novel 20q deletion. © The Author(s) 2015.

  12. Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene.

    Science.gov (United States)

    Oka, Tomoichiro; Saif, Linda J; Marthaler, Douglas; Esseili, Malak A; Meulia, Tea; Lin, Chun-Ming; Vlasova, Anastasia N; Jung, Kwonil; Zhang, Yan; Wang, Qiuhong

    2014-10-10

    The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n=7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5-6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. HOMOZYGOUS DELETION IN A SMALL-CELL LUNG-CANCER CELL-LINE INVOLVING A 3P21 REGION WITH A MARKED INSTABILITY IN YEAST ARTIFICIAL CHROMOSOMES

    NARCIS (Netherlands)

    KOK, K; van den Berg, Anke; VELDHUIS, PMJF; VANDERVEEN, AY; FRANKE, M; SCHOENMAKERS, EFPM; HULSBEEK, MMF; VANDERHOUT, AH; DELEIJ, L; VANDEVEN, W; BUYS, CHCM

    1994-01-01

    All types of lung carcinoma are characterized by a high frequency of loss of sequences from the short arm of chromosome 3, the smallest region of overlap containing D3F15S2 in band p21. Here we characterize a 440-kilobase segment from this region, which we found homozygously deleted in one of our

  14. Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits

    Directory of Open Access Journals (Sweden)

    Nicholas D. James

    2017-07-01

    Full Text Available In a recent clinical report, return of the tendon stretch reflex was demonstrated after spinal cord surgery in a case of total traumatic brachial plexus avulsion injury. Peripheral nerve grafts had been implanted into the spinal cord to reconnect to the peripheral nerves for motor and sensory function. The dorsal root ganglia (DRG containing the primary sensory nerve cells had been surgically removed in order for secondary or spinal cord sensory neurons to extend into the periphery and replace the deleted DRG neurons. The present experimental study uses a rat injury model first to corroborate the clinical finding of a re-established spinal reflex arch, and second, to elucidate some of the potential mechanisms underlying these findings by means of morphological, immunohistochemical, and electrophysiological assessments. Our findings indicate that, after spinal cord surgery, the central nervous system sensory system could replace the traumatically detached original peripheral sensory connections through new neurite growth from dendrites.

  15. Birt-Hogg-Dubé syndrome: novel FLCN frameshift deletion in daughter and father with renal cell carcinomas.

    Science.gov (United States)

    Näf, Ernst; Laubscher, Dominik; Hopfer, Helmut; Streit, Markus; Matyas, Gabor

    2016-01-01

    Germline mutation of the FLCN gene causes Birt-Hogg-Dubé syndrome (BHD), a rare autosomal dominant condition characterized by skin fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal tumours. We identified a hitherto unreported pathogenic FLCN frameshift deletion c.563delT (p.Phe188Serfs*35) in a family of a 46-year-old woman presented with macrohematuria due to bilateral chromophobe renal carcinomas. A heritable renal cancer was suspected due to the bilaterality of the tumour and as the father of this woman had suffered from renal cancer. Initially, however, BHD was overlooked by the medical team despite the highly suggestive clinical presentation. We assume that BHD is underdiagnosed, at least partially, due to low awareness of this variable condition and to insufficient use of appropriate genetic testing. Our study indicates that BHD and FLCN testing should be routinely considered in patients with positive family or personal history of renal tumours. In addition, we demonstrate how patients and their families can play a driving role in initiating genetic diagnosis, presymptomatic testing of at-risk relatives, targeted disease management, and genetic counselling of rare diseases such as BHD.

  16. Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses

    Directory of Open Access Journals (Sweden)

    María Pía Holgado

    2016-05-01

    Full Text Available MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R; or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R. The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b+/IFN-γ+ and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.

  17. Deletion of the NR4A nuclear receptor NOR1 in hematopoietic stem cells reduces inflammation but not abdominal aortic aneurysm formation.

    Science.gov (United States)

    Qing, Hua; Jones, Karrie L; Heywood, Elizabeth B; Lu, Hong; Daugherty, Alan; Bruemmer, Dennis

    2017-10-18

    The NR4A3 orphan nuclear hormone receptor, NOR1, functions as a constitutively active transcription factor to regulate inflammation, proliferation, and cell survival during pathological vascular remodeling. Inflammatory processes represent key mechanisms leading to abdominal aortic aneurysm (AAA) formation. However, a role of NOR1 in AAA formation has not been investigated previously. Inflammatory gene expression was analyzed in bone marrow-derived macrophages isolated from NOR1-deficient mice. Low-density lipoprotein receptor-deficient (LDLr -/- ) mice were irradiated and reconstituted with hematopoietic stem cells obtained from NOR1-/- or wild-type littermate mice. Animals were infused with angiotensin II and fed a diet enriched in saturated fat to induce AAA formation. Quantification of AAA formation was performed by ultrasound and ex vivo measurements. Among 184 inflammatory genes that were analyzed, 36 genes were differentially regulated in LPS-treated NOR1-deficient macrophages. Albeit this difference in gene regulation, NOR1-deficiency in hematopoietic stem cells did not affect development of AAA formation in bone marrow-derived stem cell transplanted LDLr-deficient mice. NOR1 deletion induced differential inflammatory gene transcription in macrophages but did not influence AAA formation in mice.

  18. Partial deletion 11q

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Tommerup, N; Sørensen, F B

    1995-01-01

    We describe the cytogenetic findings and the dysmorphic features in a stillborn girl with a large de novo terminal deletion of the long arm of chromosome 11. The karyotype was 46,XX,del(11)(q21qter). By reviewing previous reports of deletion 11q, we found that cleft lip and palate are most...

  19. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Ryo, Yeikou

    1975-01-01

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  20. Schizophrenia and chromosomal deletions

    Energy Technology Data Exchange (ETDEWEB)

    Lindsay, E.A.; Baldini, A. [Baylor College of Medicine, Houston, TX (United States); Morris, M. A. [Univ. of Geneva School of Medicine, NY (United States)] [and others

    1995-06-01

    Recent genetic linkage analysis studies have suggested the presence of a schizophrenia locus on the chromosomal region 22q11-q13. Schizophrenia has also been frequently observed in patients affected with velo-cardio-facial syndrome (VCFS), a disorder frequently associated with deletions within 22q11.1. It has been hypothesized that psychosis in VCFS may be due to deletion of the catechol-o-methyl transferase gene. Prompted by these observations, we screened for 22q11 deletions in a population of 100 schizophrenics selected from the Maryland Epidemiological Sample. Our results show that there are schizophrenic patients carrying a deletion of 22q11.1 and a mild VCFS phenotype that might remain unrecognized. These findings should encourage a search for a schizophrenia-susceptibility gene within the deleted region and alert those in clinical practice to the possible presence of a mild VCFS phenotype associated with schizophrenia. 9 refs.

  1. Distinct fates of self-specific T cells developing in irradiation bone marrow chimeras: Clonal deletion, clonal anergy, or in vitro responsiveness to self-Mls-1a controlled by hemopoietic cells in the thymus

    International Nuclear Information System (INIS)

    Speiser, D.E.; Chvatchko, Y.; Zinkernagel, R.M.; MacDonald, H.R.

    1990-01-01

    Elimination of potentially self-reactive T lymphocytes during their maturation in the thymus has been shown to be a major mechanism in accomplishing self-tolerance. Previous reports demonstrated that clonal deletion of self-Mls-1a-specific V beta 6+ T lymphocyte is controlled by a radiosensitive I-E+ thymic component. Irradiation chimeras reconstituted with I-E- bone marrow showed substantial numbers of mature V beta 6+ T cells despite host Mls-1a expression. Analysis of the functional properties of such chimeric T cells revealed a surprising variability in their in vitro reactivity to host Mls-1a, depending on the H-2 haplotype of stem cells used for reconstitution. In chimeras reconstituted with B10.S (H-2s) stem cells, mature V beta 6+ lymphocytes were present but functionally anergic to host-type Mls-1a in vitro. In contrast, in chimeras reconstituted with B10.G (H-2q) bone marrow, nondeleted V beta 6+ cells were highly responsive to Mls-1a in vitro. These findings suggest that clonal anergy of V beta 6+ cells to self-Mls-1a may be controlled by the affinity/avidity of T cell receptor interactions with bone marrow-derived cells in the thymus depending on the major histocompatibility complex class II molecules involved. Furthermore, chimeras bearing host (Mls-1a)-reactive V beta 6+ cells did not differ clinically from those with anergic or deleted V beta 6+ cells and survived more than one year without signs of autoimmune disease. Interestingly, their spleen cells had no Mls-1a stimulatory capacity in vitro. Therefore, regulation at the level of antigen presentation may be an alternative mechanism for maintenance of tolerance to certain self-antigens such as Mls-1a

  2. Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells

    International Nuclear Information System (INIS)

    Zheng Changyu; O'Connell, Brian C.; Baum, Bruce J.

    2003-01-01

    Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event

  3. Characterization of five partial deletions of the factor VIII gene

    International Nuclear Information System (INIS)

    Youssoufian, H.; Antonarakis, S.E.; Aronis, S.; Tsiftis, G.; Phillips, D.G.; Kazazian, H.H. Jr.

    1987-01-01

    Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, the authors have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes

  4. Quantum deletion: Beyond the no-deletion principle

    International Nuclear Information System (INIS)

    Adhikari, Satyabrata

    2005-01-01

    Suppose we are given two identical copies of an unknown quantum state and we wish to delete one copy from among the given two copies. The quantum no-deletion principle restricts us from perfectly deleting a copy but it does not prohibit us from deleting a copy approximately. Here we construct two types of a 'universal quantum deletion machine' which approximately deletes a copy such that the fidelity of deletion does not depend on the input state. The two types of universal quantum deletion machines are (1) a conventional deletion machine described by one unitary operator and (2) a modified deletion machine described by two unitary operators. Here it is shown that the modified deletion machine deletes a qubit with fidelity 3/4, which is the maximum limit for deleting an unknown quantum state. In addition to this we also show that the modified deletion machine retains the qubit in the first mode with average fidelity 0.77 (approx.) which is slightly greater than the fidelity of measurement for two given identical states, showing how precisely one can determine its state [S. Massar and S. Popescu, Phys. Rev. Lett. 74, 1259 (1995)]. We also show that the deletion machine itself is input state independent, i.e., the information is not hidden in the deleting machine, and hence we can delete the information completely from the deletion machine

  5. Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

    Science.gov (United States)

    Bianchi, Roberta; Russo, Erica; Bachmann, Samia B; Proulx, Steven T; Sesartic, Marko; Smaadahl, Nora; Watson, Steve P; Buckley, Christopher D; Halin, Cornelia; Detmar, Michael

    2017-01-01

    The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. We generated an inducible, lymphatic-specific podoplanin knockout mouse model (Pdpn ΔLEC ) and induced gene deletion postnatally. Pdpn ΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, Pdpn ΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in Pdpn ΔLEC mice. These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes. © 2016 American Heart Association, Inc.

  6. Undifferentiated embryonic cell transcription factor 1 (UTF1) and deleted in azoospermia-like (DAZL) expression in the testes of donkeys.

    Science.gov (United States)

    Lee, Y S; Jung, H J; Yoon, M J

    2017-04-01

    Putative markers for each specific germ cell stage can be a useful tool to study the fate and functions of these cells. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans, rats and horses. The deleted in azoospermia-like (DAZL) protein is also expressed by differentiated spermatogonia and primary spermatocytes in several species. However, whether the expression patterns of these molecular markers are identical and applicable to donkeys remains to be elucidated. The objective of this study was to investigate the expression patterns of UTF1 and DAZL in donkey testicular tissue, using immunohistochemistry (IHC). Testicular samples were collected from routine field castration of donkeys in Korea. The reproductive stages (pre- or post-puberty) of the testes were determined from the morphological characteristics of cross-sections of the seminiferous tubules. For IHC, the UTF1 and DAZL primary antibodies were diluted at 1:100 and 1:200, respectively. The immunolabelling revealed that UTF1 was expressed in approximately 50% of spermatogonia in the pre-pubertal stage, whereas its expression was limited to an early subset of spermatogonia in the post-pubertal stage. DAZL was expressed in some, but not all, spermatogonia in the pre-pubertal spermatogonia, and interestingly, its expression was also observed in spermatogonia and primary spermatocytes in the post-pubertal stage. Co-immunolabelling of the germ cells with both UTF1 and DAZL revealed three types of protein expression patterns at both reproductive stages, namely UTF1 only, DAZL only and both UTF1 and DAZL. These protein molecules were not expressed in Sertoli and Leydig cells. In conclusion, a co-immunolabelling system with UTF1 and DAZL antibodies may be used to identify undifferentiated (UTF1 only), differentiating (UTF1 and DAZL), and differentiated spermatogonia (DAZL only) in donkey testes. © 2017 Blackwell Verlag GmbH.

  7. Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma.

    Science.gov (United States)

    Liu, Qingqing; Cornejo, Kristine M; Cheng, Liang; Hutchinson, Lloyd; Wang, Mingsheng; Zhang, Shaobo; Tomaszewicz, Keith; Cosar, Ediz F; Woda, Bruce A; Jiang, Zhong

    2018-04-01

    Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n = 33; RO, n = 32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

    Science.gov (United States)

    Steinberg, M H; Coleman, M B; Adams, J G; Hartmann, R C; Saba, H; Anagnou, N P

    1986-02-01

    A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

  9. Deletion of SHP-2 in mesenchymal stem cells causes growth retardation, limb and chest deformity, and calvarial defects in mice

    Directory of Open Access Journals (Sweden)

    Philip E. Lapinski

    2013-11-01

    In mice, induced global disruption of the Ptpn11 gene, which encodes the SHP-2 tyrosine phosphatase, results in severe skeletal abnormalities. To understand the extent to which skeletal abnormalities can be attributed to perturbation of SHP-2 function in bone-forming osteoblasts and chondrocytes, we generated mice in which disruption of Ptpn11 is restricted to mesenchymal stem cells (MSCs and their progeny, which include both cell types. MSC-lineage-specific SHP-2 knockout (MSC SHP-2 KO mice exhibited postnatal growth retardation, limb and chest deformity, and calvarial defects. These skeletal abnormalities were associated with an absence of mature osteoblasts and massive chondrodysplasia with a vast increase in the number of terminally differentiated hypertrophic chondrocytes in affected bones. Activation of mitogen activated protein kinases (MAPKs and protein kinase B (PKB; also known as AKT was impaired in bone-forming cells of MSC SHP-2 KO mice, which provides an explanation for the skeletal defects that developed. These findings reveal a cell-autonomous role for SHP-2 in bone-forming cells in mice in the regulation of skeletal development. The results add to our understanding of the pathophysiology of skeletal abnormalities observed in humans with germline mutations in the PTPN11 gene (e.g. Noonan syndrome and LEOPARD syndrome.

  10. Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice.

    Directory of Open Access Journals (Sweden)

    Jesus Gil-Pulido

    Full Text Available Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3 is required for the development of classical CD8α+ and CD103+ DCs. By crossing mice deficient in Batf3 with atherosclerosis-prone low density lipoprotein receptor (Ldlr-/--deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α+ and CD103+ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.

  11. Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice.

    Science.gov (United States)

    Lan, Zi-Jian; Krause, M S; Redding, S D; Li, X; Wu, G Z; Zhou, H X; Bohler, H C; Ko, C; Cooney, A J; Zhou, Junmei; Lei, Z M

    2017-03-15

    Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Enhanced genome editing tools for multi-gene deletion knock-out approaches using paired CRISPR sgRNAs in CHO cells

    DEFF Research Database (Denmark)

    Schmieder, Valerie; Bydlinski, Nina; Strasser, Richard

    2017-01-01

    (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by deleting the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. Fut8 also served as model...

  13. Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells

    International Nuclear Information System (INIS)

    Ayusawa, D.; Koyama, H.; Shimizu, K.; Kaneda, S.; Takeishi, K.; Seno, T.

    1986-01-01

    Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells

  14. Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

    Science.gov (United States)

    Geber, A; Hitchcock, C A; Swartz, J E; Pullen, F S; Marsden, K E; Kwon-Chung, K J; Bennett, J E

    1995-01-01

    We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae. PMID:8593007

  15. Single and combined deletions of the NTAL/LAB and LAT adaptors minimally affect B-cell development and function

    Czech Academy of Sciences Publication Activity Database

    Wang, Y.; Horváth, Ondřej; Hamm-Baarke, A.; Richelme, M.; Grégoire, C.; Guinamard, R.; Hořejší, Václav; Angelisová, Pavla; Špička, Jiří; Schraven, B.; Malissen, B.; Malissen, M.

    2005-01-01

    Roč. 25, č. 11 (2005), s. 4455-4465 ISSN 0270-7306 R&D Projects: GA MŠk(CZ) 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : NTAL * LAT * B-cell Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.093, year: 2005

  16. Reduced host cell invasiveness and oxidative stress tolerance in double and triple csp gene family deletion mutants of Listeria monocytogenes.

    Science.gov (United States)

    Loepfe, Chantal; Raimann, Eveline; Stephan, Roger; Tasara, Taurai

    2010-07-01

    The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and

  17. ET-1 deletion from endothelial cells protects the kidney during the extension phase of ischemia/reperfusion injury

    Energy Technology Data Exchange (ETDEWEB)

    Arfian, Nur [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Emoto, Noriaki, E-mail: emoto@med.kobe-u.ac.jp [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Vignon-Zellweger, Nicolas; Nakayama, Kazuhiko; Yagi, Keiko [Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Hirata, Ken-ichi [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Ischemia/reperfusion injury (IRI) induced increased endothelin-1 (ET-1) expression. Black-Right-Pointing-Pointer IRI was accompanied by tubular injury and remodeling of renal arteries. Black-Right-Pointing-Pointer IRI increased oxidative stress and inflammation. Black-Right-Pointing-Pointer Genetic suppression of ET-1 in endothelial cells attenuates IRI in the kidney. Black-Right-Pointing-Pointer The mechanisms include the inhibition of oxidative stress and inflammation. -- Abstract: Background: The prognosis of patients after acute kidney injury (AKI) is poor and treatment is limited. AKI is mainly caused by renal ischemia/reperfusion injury (IRI). During the extension phase of IRI, endothelial damage may participate in ischemia and inflammation. Endothelin-1 (ET-1) which is mostly secreted by endothelial cells is an important actor of IRI, particularly through its strong vasoconstrictive properties. We aimed to analyze the specific role of ET-1 from the endothelial cells in AKI. Methods: We used mice lacking ET-1 in the vascular endothelial cells (VEETKO). We induced IRI in VEETKO mice and wild type controls by clamping both kidneys for 30 min. Sham operated mice were used as controls. Mice were sacrificed one day after IRI in order to investigate the extension phase of IRI. Kidney function was assessed based on serum creatinine concentration. Levels of expression of ET-1, its receptor ET{sub A}, protein kinase C, eNOS, E-Cadherin and inflammation markers were evaluated by real time PCR or western blot. Tubular injury was scored on periodic acid Schiff stained kidney preparations. Lumen and wall area of small intrarenal arteries were measured on kidney slices stained for alpha smooth muscle cell actin. Oxidative stress, macrophage infiltration and cell proliferation was evaluated on slices stained for 8-hydroxy-2 Prime -deoxyguanosine, F4/80 and PCNA, respectively. Results: IRI induced kidney failure and increased ET-1 and

  18. Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

    International Nuclear Information System (INIS)

    Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi; Baines, Joel D.

    2004-01-01

    Previous results indicated that the herpes simplex virus 1 (HSV-1) U L 31 gene is necessary and sufficient for localization of the U L 34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U L 31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U L 31 gene. The replication of the U L 31 deletion virus was restored on U L 31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U L 34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U L 34 protein localized at the nuclear membrane in rabbit skin cells, and U L 31 complementing CV1 cells infected with the U L 31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U L 34 protein to the nuclear membrane. We speculate that this function partially complements that of U L 31 and may explain why U L 31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells

  19. The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

    Science.gov (United States)

    Saveliev, S V; Cox, M M

    1994-01-01

    Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5). Images PMID:7838724

  20. Allogeneic stem cell transplant in patients with chronic lymphocytic leukemia with 17p deletion: consult-transplant versus consult- no-transplant analysis.

    Science.gov (United States)

    Poon, Michelle L; Fox, Patricia S; Samuels, Barry I; O'Brien, Susan; Jabbour, Elias; Hsu, Yvonne; Gulbis, Alison; Korbling, Martin; Champlin, Richard; Abruzzo, Lynne V; Bassett, Roland L; Khouri, Issa F

    2015-03-01

    Allogeneic stem cell transplant (alloSCT) can overcome the adverse prognosis of chronic lymphocytic leukemia with 17p deletion (17p- CLL). However, its applicability remains unclear. Since 2007, our leukemia service has referred patients with 17p- CLL for alloSCT at presentation. In this study, the outcomes of these patients were reviewed retrospectively to determine whether they underwent alloSCT and why patients did not undergo alloSCT. Fifty-two patients with 17p- CLL who were referred to the transplant service from 2007 to 2010 were identified. Of these patients, 32 (62%) did not undergo alloSCT, mainly because of treatment- or disease-related complications (n = 15). The 2-year post-referral overall survival rates of the alloSCT and non-SCT groups were 64% and 25%, respectively (p = 0.001). These findings suggest that while alloSCT is an effective therapy in patients with 17p- CLL, pre-SCT complications may preclude a significant proportion of patients from undergoing the procedure.

  1. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

    Directory of Open Access Journals (Sweden)

    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  2. Inducible deletion of CD28 prior to secondary nippostrongylus brasiliensis infection impairs worm expulsion and recall of protective memory CD4⁺ T cell responses.

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    Hlumani Ndlovu

    2014-02-01

    Full Text Available IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28⁻/⁻ mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28(-/loxCre⁺/⁻+TM to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28⁻/⁻ mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28(-/loxCre⁺/⁻ mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28⁻/⁻ mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T(FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28⁻/⁻ mice and tamoxifen treated CD28(-/loxCre⁺/⁻ mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28(-/loxCre⁺/⁻ mice. Therefore, it can be concluded that CD28

  3. Targeted Gene Deletion Demonstrates that Cell Adhesion MoleculeICAM-4 is Critical for Erythroblastic Island Formation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gloria; Lo, Annie; Short, Sarah A.; Mankelow, Tosti J.; Spring, Frances; Parsons, Stephen F.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

    2006-02-15

    Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule-4 (ICAM-4), animmunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha V integrins as ICAM-4 counter receptors. Since macrophages express alpha V, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knockout mice and developed quantitative, live cell techniques for harvesting intact islands and for reforming islands in vitro. We observed a 47 percent decrease in islands reconstituted from ICAM-4 null marrow compared to wild type. We also found a striking decrease in islands formed in vivo in knockout mice. Further, peptides that block ICAM-4 alpha V adhesion produced a 53-57 percent decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha V functions in island integrity. Importantly, we documented that alpha V integrin is expressed in macrophages isolated from erythro blastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

  4. Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

    Science.gov (United States)

    Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

    2015-07-01

    Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

  5. Klf5 deletion promotes Pten deletion-initiated luminal-type mouse prostate tumors through multiple oncogenic signaling pathways.

    Science.gov (United States)

    Xing, Changsheng; Ci, Xinpei; Sun, Xiaodong; Fu, Xiaoying; Zhang, Zhiqian; Dong, Eric N; Hao, Zhao-Zhe; Dong, Jin-Tang

    2014-11-01

    Krüppel-like factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer-mutation/deletion of Pten and deletion of Klf5.

  6. Allogeneic hematopoietic stem-cell transplantation for chronic lymphocytic leukemia with 17p deletion: a retrospective European Group for Blood and Marrow Transplantation analysis.

    Science.gov (United States)

    Schetelig, Johannes; van Biezen, Anja; Brand, Ronald; Caballero, Dolores; Martino, Rodrigo; Itala, Maija; García-Marco, José A; Volin, Liisa; Schmitz, Norbert; Schwerdtfeger, Rainer; Ganser, Arnold; Onida, Francesco; Mohr, Brigitte; Stilgenbauer, Stephan; Bornhäuser, Martin; de Witte, Theo; Dreger, Peter

    2008-11-01

    Patients with chronic lymphocytic leukemia (CLL) and 17p deletion (17p-) have a poor prognosis. Although allogeneic hematopoietic stem-cell transplantation (HCT) has the potential to cure patients with advanced CLL, it is not known whether this holds true for patients with 17p-CLL. Baseline data from patients, for whom information on the presence of 17p-CLL was available, were downloaded from the European Group for Blood and Marrow Transplantation database. Additional information on the course of CLL and follow-up was collected with a questionnaire. A total of 44 patients with 17p-CLL received allogeneic HCT between March 1995 and July 2006 from a matched sibling (n = 24) or an alternative donor (n = 20). 17p-CLL had been diagnosed by fluorescent in situ hybridization in 82% of patients and by conventional banding in 18% of patients. The median age was 54 years. Before HCT, a median of three lines of chemotherapy had been administered. At HCT, 53% of patients were in remission. Reduced-intensity conditioning was applied in 89% of patients. Acute, grade 2 to 4 graft-versus-host disease (GVHD) occurred in 43% of patients, and extensive chronic GVHD occurred in 53% of patients. At last follow-up, 19 patients were alive, with a median observation time of 39 months (range, 18 to 101 months). Three-year overall survival and progression-free survival rates were 44% and 37%, respectively. The cumulative incidence of progressive disease at 4 years was 34%. No late relapse occurred in nine patients with a follow-up longer than 4 years. Allogeneic HCT has the potential to induce long-term disease-free survival in patients with 17p-CLL.

  7. Chemotherapy refractory testicular germ cell tumor is associated with a variant in Armadillo Repeat gene deleted in Velco-Cardio-Facial syndrome (ARVCF

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    Chunkit eFung

    2012-12-01

    Full Text Available Introduction: There is evidence that inherited genetic variation affects both testicular germ cell tumor (TGCT treatment outcome and risks of late-complications arising from cisplatin-based chemotherapy. Using a candidate gene approach, we examined associations of three genes involved in the cisplatin metabolism pathway, GSTP1, COMT, and TPMT, with TGCT outcome and cisplatin-induced neurotoxicity. Material and Methods: Our study population includes a subset of patients (n=137 from a genome-wide association study at the University of Pennsylvania that evaluates inherited genetic susceptibility to TGCT. All patients in our study had at least one course of cisplatin-based chemotherapy with at least one year of follow up. A total of 90 markers in GSTP1, COMT and TPMT and their adjacent genomic regions (± 20 kb were analyzed for associations with refractory TGCT after first course of chemotherapy, progression-free survival (PFS, overall survival (OS, peripheral neuropathy, and ototoxicity. Results: After adjustment for multiple comparisons, one SNP, rs2073743, in the flanking region (± 20 kb of COMT was associated with refractory TGCT after initial chemotherapy. This SNP lies within the intron region of the Armadillo Repeat gene deleted in Velco-Cardio-Facial syndrome (ARVCF. The G allele of rs2073743 predisposed patients to refractory disease with a relative risk of 2.6 (95% CI 1.1, 6.3; P=0.03. Assuming recessive inheritance, patients with the GG genotype had 22.7 times higher risk (95% CI 3.3, 155.8; P=0.04 of developing refractory disease when compared to those with the GC or CC genotypes. We found no association of our candidate genes with peripheral neuropathy, ototoxicity, PFS and OS. Discussion: This is the first study to suggest that germline genetic variants of ARVCF may affect TGCT outcome. The result of this study is hypothesis generating and should be validated in future studies.

  8. Frequent deletion of 3p21.1 region carrying semaphorin 3G and aberrant expression of the genes participating in semaphorin signaling in the epithelioid type of malignant mesothelioma cells.

    Science.gov (United States)

    Yoshikawa, Yoshie; Sato, Ayuko; Tsujimura, Tohru; Morinaga, Tomonori; Fukuoka, Kazuya; Yamada, Shusai; Murakami, Aki; Kondo, Nobuyuki; Matsumoto, Seiji; Okumura, Yoshitomo; Tanaka, Fumihiro; Hasegawa, Seiki; Hashimoto-Tamaoki, Tomoko; Nakano, Takashi

    2011-12-01

    Array-based comparative genomic hybridization analysis was performed on 21 malignant mesothelioma (MM) samples (16 primary cell cultures and 5 cell lines) and two reactive mesothelial hyperplasia (RM) primary cell cultures. The RM samples did not have any genomic losses or gains. In MM samples, deletions in 1p, 3p21, 4q, 9p21, 16p13 and 22q were detected frequently. We focused on 3p21 because this deletion was specific to the epithelioid type. Especially, a deletion in 3p21.1 region carrying seven genes including SEMA3G was found in 52% of MM samples (11 of 14 epithelioid samples). The allele loss of 3p21.1 might be a good marker for the epithelioid MM. A homozygous deletion in this region was detected in two MM primary cell cultures. A heterozygous deletion detected in nine samples contained the 3p21.1 region and 3p21.31 one carrying the candidate tumor suppressor genes such as semaphorin 3F (SEMA3F), SEMA3B and Ras association (RalGDS/AF-6) domain family member 1 (RASSF1A). SEMA3B, 3F and 3G are class 3 semaphorins and inhibit growth by competing with vascular endothelial growth factor (VEGF) through binding to neuropilin. All MM samples downregulated the expression of more than one gene for SEMA3B, 3F and 3G when compared with Met5a, a normal pleura-derived cell line. Moreover, in 12 of 14 epithelioid MM samples the expression level of SEMA3A was lower than that in Met5a and the two RM samples. An augmented expression of VEGFA was detected in half of the MM samples. The expression ratio of VEGFA/SEMA3A was significantly higher in the epithelioid MMs than in Met5a, RMs and the non-epithelioid MMs. Our data suggest that the downregulated expression of SEMA3A and several SEMA3s results in a loss of inhibitory activities in tumor angiogenesis and tumor growth of VEGFA; therefore, it may play an important role on the pathogenesis of the epithelioid type of MM.

  9. Interstitial granulomatous dermatitis (IGD)

    NARCIS (Netherlands)

    Tebeica, Tiberiu; Voicu, Cristiana; Patterson, James W.; Mangarov, Hristo; Lotti, T.; Wollina, Uwe; Lotti, Jacopo; França, Katlein; Batashki, Atanas; Tchernev, Georgi

    2017-01-01

    We report the case of a 42 years old male patient suffering from skin changes, which appeared in the last 7-8 years. Two biopsies were performed during the evolution of the lesion. Both showed similar findings that consisted in a busy dermis with interstitial, superficial and deep infiltrates of

  10. Conditional deletion of Pten causes bronchiolar hyperplasia.

    Science.gov (United States)

    Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

    2008-03-01

    Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

  11. Dendritic cell-specific deletion of β-catenin results in fewer regulatory T-cells without exacerbating autoimmune collagen-induced arthritis

    NARCIS (Netherlands)

    C.H. Alves (Celso Henrique); J.L. Ober-Blöbaum (Julia); I. Brouwers-Haspels (Inge); P. Asmawidjaja (Patrick); A.M.C. Mus (Adriana); W. Razawy (Wida); M. Molendijk (Marlieke); B.E. Clausen (Bjorn); E.W. Lubberts (Erik)

    2015-01-01

    textabstractDendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote

  12. Deletions of 16q in Wilms tumors localize to blastemal-anaplastic cells and are associated with reduced expression of the IRXB renal tubulogenesis gene cluster

    NARCIS (Netherlands)

    Mengelbier, Linda Holmquist; Karlsson, Jenny; Lindgren, David; Øra, Ingrid; Isaksson, Margareth; Frigyesi, Ildiko; Frigyesi, Attila; Bras, Johannes; Sandstedt, Bengt; Gisselsson, David

    2010-01-01

    Wilms tumor is the most common pediatric renal neoplasm, but few molecular prognostic markers have been identified for this tumor. Somatic deletion in the long arm of chromosome 16 (16q) is known to predict a less favorable outcome in Wilms tumor, but the underlying molecular mechanisms are not

  13. Selective silencing of full-length CD80 but not IgV-CD80 leads to impaired clonal deletion of self-reactive T cells and altered regulation of immune responses.

    Science.gov (United States)

    Bugeon, L; Hargreaves, R E; Crompton, T; Outram, S; Rahemtulla, A; Porter, A C; Dallman, M J

    2001-01-01

    Co-stimulation provided by the B7 family of proteins underpins the development of protective immunity. There are three identified members of this family: CD80, its splice variant IgV-CD80 and CD86. It has hitherto been difficult to analyze the expression and function of IgV-CD80 since there are no appropriate reagents capable of distinguishing it from CD80. We have generated mice, by gene targeting, the lack CD80 whilst maintaining expression of IgV-CD80. Mutant animals did not delete T cells bearing mammary tumor virus-reactive TCR as efficiently as wild-type animals. We also demonstrate the importance of IgV-CD80 in the responses of recently activated cells and reveal a role for CD80 in sustaining T cell responses. CD86, whilst critical to primary T cell activation, made only a minor contribution to re-activation of normal cells.

  14. The effect of miR-338-3p on HBx deletion-mutant (HBx-d382 mediated liver-cell proliferation through CyclinD1 regulation.

    Directory of Open Access Journals (Sweden)

    Xiaoyu Fu

    Full Text Available Hepatitis B Virus (HBV DNA integration and HBV X (HBx deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382-400 can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3'UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells, and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3'UTR region, the effect of miR-338-3p on the second binding site (nt 2397-2403 was required for the inhibition.miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3'UTR region, mainly at nt 2397-2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the

  15. A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

    Science.gov (United States)

    Retamal-Díaz, Angello R.; Kalergis, Alexis M.; Bueno, Susan M.; González, Pablo A.

    2017-01-01

    Herpes simplex virus type 2 (HSV-2) is highly prevalent in the human population producing significant morbidity, mainly because of the generation of genital ulcers and neonatal encephalitis. Additionally, HSV-2 infection significantly increases the susceptibility of the host to acquire HIV and promotes the shedding of the latter in the coinfected. Despite numerous efforts to create a vaccine against HSV-2, no licensed vaccines are currently available. A long-standing strategy, based on few viral glycoproteins combined with adjuvants, recently displayed poor results in a Phase III clinical study fueling exploration on the development of mutant HSV viruses that are attenuated in vivo and elicit protective adaptive immune components, such as antiviral antibodies and T cells. Importantly, such specialized antiviral immune components are likely induced and modulated by dendritic cells, professional antigen presenting cells that process viral antigens and present them to T cells. However, HSV interferes with several functions of DCs and ultimately induces their death. Here, we propose that for an attenuated mutant virus to confer protective immunity against HSV in vivo based on adaptive immune components, such virus should also be attenuated in dendritic cells to promote a robust and effective antiviral response. We provide a background framework for this idea, considerations, as well as the means to assess this hypothesis. Addressing this hypothesis may provide valuable insights for the development of novel, safe, and effective vaccines against herpes simplex viruses. PMID:28848543

  16. A Herpes Simplex Virus Type 2 Deleted for Glycoprotein D Enables Dendritic Cells to Activate CD4+ and CD8+ T Cells

    Directory of Open Access Journals (Sweden)

    Angello R. Retamal-Díaz

    2017-08-01

    Full Text Available Herpes simplex virus type 2 (HSV-2 is highly prevalent in the human population producing significant morbidity, mainly because of the generation of genital ulcers and neonatal encephalitis. Additionally, HSV-2 infection significantly increases the susceptibility of the host to acquire HIV and promotes the shedding of the latter in the coinfected. Despite numerous efforts to create a vaccine against HSV-2, no licensed vaccines are currently available. A long-standing strategy, based on few viral glycoproteins combined with adjuvants, recently displayed poor results in a Phase III clinical study fueling exploration on the development of mutant HSV viruses that are attenuated in vivo and elicit protective adaptive immune components, such as antiviral antibodies and T cells. Importantly, such specialized antiviral immune components are likely induced and modulated by dendritic cells, professional antigen presenting cells that process viral antigens and present them to T cells. However, HSV interferes with several functions of DCs and ultimately induces their death. Here, we propose that for an attenuated mutant virus to confer protective immunity against HSV in vivo based on adaptive immune components, such virus should also be attenuated in dendritic cells to promote a robust and effective antiviral response. We provide a background framework for this idea, considerations, as well as the means to assess this hypothesis. Addressing this hypothesis may provide valuable insights for the development of novel, safe, and effective vaccines against herpes simplex viruses.

  17. CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

    Science.gov (United States)

    Wu, Yu-Jing; Chen, Heng-Shi; Chen, Wen-Sheng; Dong, Jin; Dong, Xiao-Jie; Dai, Xing; Huang, Qiong; Wei, Wei

    2018-01-01

    Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'- O -benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4 + T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4 + T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4 + T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr 394 ). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4 + T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr 394 ) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4 + T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

  18. CRISPR/Cas9-Mediated Deletion of C1EIS Inhibits Chicken Embryonic Stem Cell Differentiation Into Male Germ Cells (Gallus gallus).

    Science.gov (United States)

    Zuo, Qisheng; Jin, Kai; Wang, Yingjie; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2017-08-01

    We previously found that C1EIS is preferentially expressed in Chicken spermatogonial stem cells (SSCs) by RNA sequencing (RNA-seq), so our current study focused on C1EIS's role in Chicken embryonic stem cells (ESCs) differentiation into male germ cells. We constructed a CRISPR/Cas9 vector targeting C1EIS. T7 endonuclease I (T7EI) digestion method and sequencing of TA cloning were used to detect the knock-out efficiency of the Single guide RNA (sgRNA) after the cas9/gRNA vector transfected into D fibroblasts 1(DF-1), ESCs, and Chicken embryos. The results showed that CRISPR/Cas9 gene knockout efficiency is about 40%. Differentiation of the targeted ESCs into SSCs was inhibited at the embryoid body stage due to C1EIS deficiency. Immunofluorescent staining revealed that the mutagenized ESCs (RA (Retinoic Acid) with C1EIS Knock out) expressed lower levels of integrin α6 and integrin β1 compared to wild type cells. Quantitative real-time PCR (QRT-PCR) revealed Oct4 and Sox2 expression significantly increased, contrarily integrin β1 and Stra8 expression significantly decreased than RA induced group and RA with C1EIS Overexpression. During retinoic acid-induced differentiation, knockout of C1EIS in ESCs inhibited formation of SSC-like cells, suggesting C1EIS plays a vital role in promoting differentiation of avian ESCs to SSCs by regulating expression of multiple pluripotency-related genes. J. Cell. Biochem. 118: 2380-2386, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

    Science.gov (United States)

    Maier, Lisa-Katharina; Benz, Juliane; Fischer, Susan; Alstetter, Martina; Jaschinski, Katharina; Hilker, Rolf; Becker, Anke; Allers, Thorsten; Soppa, Jörg; Marchfelder, Anita

    2015-10-01

    Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. 9q22 Deletion - First Familial Case

    Directory of Open Access Journals (Sweden)

    Yamamoto Toshiyuki

    2011-06-01

    Full Text Available Abstract Background Only 29 cases of constitutional 9q22 deletions have been published and all have been sporadic. Most associate with Gorlin syndrome or nevoid basal cell carcinoma syndrome (NBCCS, MIM #109400 due to haploinsufficiency of the PTCH1 gene (MIM *601309. Methods and Results We report two mentally retarded female siblings and their cognitively normal father, all carrying a similar 5.3 Mb microdeletion at 9q22.2q22.32, detected by array CGH (244 K. The deletion does not involve the PTCH1 gene, but instead 30 other gene,s including the ROR2 gene (MIM *602337 which causing both brachydactyly type 1 (MIM #113000 and Robinow syndrome (MIM #268310, and the immunologically active SYK gene (MIM *600085. The deletion in the father was de novo and FISH analysis of blood lymphocytes did not suggest mosaicism. All three patients share similar mild dysmorphic features with downslanting palpebral fissures, narrow, high bridged nose with small nares, long, deeply grooved philtrum, ears with broad helix and uplifted lobuli, and small toenails. All have significant dysarthria and suffer from continuous middle ear and upper respiratory infections. The father also has a funnel chest and unilateral hypoplastic kidney but the daughters have no malformations. Conclusions This is the first report of a familial constitutional 9q22 deletion and the first deletion studied by array-CGH which does not involve the PTCH1 gene. The phenotype and penetrance are variable and the deletion found in the cognitively normal normal father poses a challenge in genetic counseling.

  1. The significance of chromosome deletions in atomic-bomb survivors

    International Nuclear Information System (INIS)

    Tanaka, Kimio; Shigeta, Chiharu; Oguma, Nobuo; Kamada, Nanao; Deng, Z.; Niimi, Masanobu; Aisaka, Tadaichi.

    1986-01-01

    In 39 A-bomb survivors 40 years after exposure at ≤ 1,000 m from ground zero, the frequency and features of chromosome deletions in peripheral lymphocytes were examined using a differential staining technique. Simultaneously, in vitro irradiation experiment with Cf-252 was made to infer chromosome aberrations occuring immediately after exposure. Californium-252 with 100 rad induced dicentric and ring chromosomes in 40 % of the cells and acentric fragments in 44 %. Among the A-bomb survivors, chromosome aberrations were observed in 651 (21 %) of the total 3,136 cells. There were 146 cells with deletions (22 % of abnormal cells; 5 % of the total cells), and 10 cells with acentric fragment (0.3 % of the total cells). The figure for deletions was far higher than that reported in the literature. A large number of deletions were seen in chromosomes no.4, no.21, and no.22, and a few deletions in chromosomes no.7 and no.20. Significance of chromosome deletions is discussed. (Namekawa, K.)

  2. Attenuation of monkeypox virus by deletion of genomic regions

    Science.gov (United States)

    Lopera, Juan G.; Falendysz, Elizabeth A.; Rocke, Tonie E.; Osorio, Jorge E.

    2015-01-01

    Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivostudies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.

  3. CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

    Directory of Open Access Journals (Sweden)

    Kentaro Tsukamoto

    Full Text Available Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9 system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

  4. DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1

    International Nuclear Information System (INIS)

    Lerner, Mikael; Harada, Masako; Loven, Jakob; Castro, Juan; Davis, Zadie; Oscier, David; Henriksson, Marie; Sangfelt, Olle; Grander, Dan; Corcoran, Martin M.

    2009-01-01

    The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

  5. Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Carl Owen

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B, a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/- were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/- mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD-fed adip-crePTP1B(-/- mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.

  6. On Deletion of Sutra Translation

    Institute of Scientific and Technical Information of China (English)

    CHEN Shu-juan

    2017-01-01

    Dao An's the metaphor of translation "wine diluted with water' ' expressed a view about translation that had been abridged.Later Kumarajiva provided metaphor "rice chewed—tasteless and downright disgusting".Both of them felt regretted at the weakening of taste,sometimes even the complete loss of flavor caused by deletion in translation of Buddhist sutras.In early sutra translation,deletion is unavoidable which made many sutra translators felt confused and drove them to study it further and some even managed to give their understanding to this issue.This thesis will discuss the definition,and what causes deletion and the measures adopted by the sutra translators.

  7. Generation of induced pluripotent stem cells from a Becker muscular dystrophy patient carrying a deletion of exons 45-55 of the dystrophin gene (CCMi002BMD-A-9 ∆45-55

    Directory of Open Access Journals (Sweden)

    Aoife Gowran

    2018-04-01

    Full Text Available Becker muscular dystrophy (BMD is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55.

  8. Generation of induced pluripotent stem cells from a Becker muscular dystrophy patient carrying a deletion of exons 45-55 of the dystrophin gene (CCMi002BMD-A-9 ∆45-55).

    Science.gov (United States)

    Gowran, Aoife; Spaltro, Gabriella; Casalnuovo, Federica; Vigorelli, Vera; Spinelli, Pietro; Castiglioni, Elisa; Rovina, Davide; Paganini, Stefania; Di Segni, Marina; Gervasini, Cristina; Nigro, Patrizia; Pompilio, Giulio

    2018-04-01

    Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55). Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2007-08-01

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

  10. Extensive diversification of IgD-, IgY-, and truncated IgY(δFc)-encoding genes in the red-eared turtle (Trachemys scripta elegans).

    Science.gov (United States)

    Li, Lingxiao; Wang, Tao; Sun, Yi; Cheng, Gang; Yang, Hui; Wei, Zhiguo; Wang, Ping; Hu, Xiaoxiang; Ren, Liming; Meng, Qingyong; Zhang, Ran; Guo, Ying; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

    2012-10-15

    IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single μ gene was identified, the α gene was not identified in this species.

  11. Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

    DEFF Research Database (Denmark)

    Jensen, L T; Ladefoged, S; Birkelund, S

    1995-01-01

    characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells...

  12. Strategies for state-dependent quantum deleting

    International Nuclear Information System (INIS)

    Song Wei; Yang Ming; Cao Zhuoliang

    2004-01-01

    A quantum state-dependent quantum deleting machine is constructed. We obtain a upper bound of the global fidelity on N-to-M quantum deleting from a set of K non-orthogonal states. Quantum networks are constructed for the above state-dependent quantum deleting machine when K=2. Our deleting protocol only involves a unitary interaction among the initial copies, with no ancilla. We also present some analogies between quantum cloning and deleting

  13. Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells

    DEFF Research Database (Denmark)

    Müller, Hanna; Nagel, Christian; Weiss, Christel

    2015-01-01

    between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well...... as with DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6...... that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker....

  14. Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

    Science.gov (United States)

    Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

    2016-01-01

    ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

  15. ATLAS DQ2 Deletion Service

    International Nuclear Information System (INIS)

    Oleynik, Danila; Petrosyan, Artem; Garonne, Vincent; Campana, Simone

    2012-01-01

    The ATLAS Distributed Data Management project DQ2 is responsible for the replication, access and bookkeeping of ATLAS data across more than 100 distributed grid sites. It also enforces data management policies decided on by the collaboration and defined in the ATLAS computing model. The DQ2 Deletion Service is one of the most important DDM services. This distributed service interacts with 3rd party grid middleware and the DQ2 catalogues to serve data deletion requests on the grid. Furthermore, it also takes care of retry strategies, check-pointing transactions, load management and fault tolerance. In this paper special attention is paid to the technical details which are used to achieve the high performance of service, accomplished without overloading either site storage, catalogues or other DQ2 components. Special attention is also paid to the deletion monitoring service that allows operators a detailed view of the working system.

  16. Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells.

    Directory of Open Access Journals (Sweden)

    Roman Covarrubias

    Full Text Available Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr on antigen presenting cells (APCs has been shown to enhance invariant natural killer T (iNKT cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP, plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ. We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO. LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

  17. Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

    Science.gov (United States)

    Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

    2015-12-01

    Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research.

  18. Nature of mutants induced by ionizing radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by. gamma. -rays or. cap alpha. -particles showing that the majority have deletions of all or part of the hprt gene

    Energy Technology Data Exchange (ETDEWEB)

    Thacker, J

    1986-05-01

    DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionizing radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the ..gamma..-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. 16 references, 4 figures, 1 table.

  19. Clinicopathological and Targeted Exome Gene Features of a Patient with Metastatic Acinic Cell Carcinoma of the Parotid Gland Harboring an ARID2 Nonsense Mutation and CDKN2A/B Deletion

    Directory of Open Access Journals (Sweden)

    Wayne A. Warner

    2015-01-01

    Full Text Available We describe the presentation, treatment, clinical outcome, and targeted genome analysis of a metastatic salivary acinic cell carcinoma (AciCC. A 71-year-old male presented with a 3 cm right tail of a parotid lesion, first detected as a nodule by the patient seven months earlier. He had a right total parotidectomy with cranial nerve VII resection, right facial nerve resection and grafting, resection of the right conchal cartilage, and right modified radical neck dissection. The primary tumor revealed AciCC with two distinct areas: a well-differentiated component with glandular architecture and a dedifferentiated component with infiltrative growth pattern associated with prominent stromal response, necrosis, perineural invasion, and cellular pleomorphism. Tumor staging was pT4 N0 MX. Immunohistochemistry staining showed pankeratin (+, CD56 (−, and a Ki67 proliferation index of 15%. Upon microscopic inspection, 49 local lymph nodes resected during parotidectomy were negative for cancer cells. Targeted sequencing of the primary tumor revealed deletions of CDKN2A and CDKN2B, a nonsense mutation in ARID2, and single missense mutations of unknown significance in nine other genes. Despite postoperative localized radiation treatment, follow-up whole body PET/CT scan showed lung, soft tissue, bone, and liver metastases. The patient expired 9 months after resection of the primary tumor.

  20. Chemoimmunotherapy for Relapsed/Refractory and Progressive 17p13 Deleted Chronic Lymphocytic Leukemia (CLL) Combining Pentostatin, Alemtuzumab, and Low Dose Rituximab is Effective and Tolerable and Limits Loss of CD20 Expression by Circulating CLL Cells

    Science.gov (United States)

    Zent, Clive S.; Taylor, Ronald P.; Lindorfer, Margaret A.; Beum, Paul V.; LaPlant, Betsy; Wu, Wenting; Call, Timothy G.; Bowen, Deborah A.; Conte, Michael J.; Frederick, Lori A.; Link, Brian K.; Blackwell, Sue E.; Veeramani, Suresh; Baig, Nisar A.; Viswanatha, David S.; Weiner, George J.; Witzig, Thomas E.

    2014-01-01

    Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analogue refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a phase II trial testing the efficacy and tolerability of a short duration regimen combining pentostatin, alemtuzumab, and low dose high frequency rituximab (PAR) designed to decrease the risk of treatment associated infections and limit loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n=36) or previously untreated with 17p13 deletion (17p13-)(n=3). Thirteen (33%) patients had both 17p13- and TP53 mutations predicted to be dysfunctional and eight patients had purine analogue refractory CLL without TP53 dysfunction. Twenty-six (67%) patients completed therapy with only five (13%) patients having treatment limiting toxicity, and no treatment related deaths. Twenty-two (56%) patients responded to treatment with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression free survival was 7.2 months, time to next treatment 9.1 months, and overall survival 34.1 months. The majority of deaths (82%) were caused by progressive disease including transformed diffuse large B cell lymphoma (n=6). Correlative studies showed that low dose rituximab activates complement and NK cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that PAR is a tolerable and effective therapy for CLL and that low dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression. PMID:24723493

  1. Chemoimmunotherapy for relapsed/refractory and progressive 17p13-deleted chronic lymphocytic leukemia (CLL) combining pentostatin, alemtuzumab, and low-dose rituximab is effective and tolerable and limits loss of CD20 expression by circulating CLL cells.

    Science.gov (United States)

    Zent, Clive S; Taylor, Ronald P; Lindorfer, Margaret A; Beum, Paul V; LaPlant, Betsy; Wu, Wenting; Call, Timothy G; Bowen, Deborah A; Conte, Michael J; Frederick, Lori A; Link, Brian K; Blackwell, Sue E; Veeramani, Suresh; Baig, Nisar A; Viswanatha, David S; Weiner, George J; Witzig, Thomas E

    2014-07-01

    Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab-containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short-duration regimen combining pentostatin, alemtuzumab, and low-dose high-frequency rituximab designed to decrease the risk of treatment-associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13-) (n = 3). Thirteen (33%) patients had both 17p13- and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty-six (67%) patients completed therapy, with only five (13%) patients having treatment-limiting toxicity and no treatment-related deaths. Twenty-two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression-free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B-cell lymphoma (n = 6). Correlative studies showed that low-dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low-dose high-frequency rituximab is a tolerable and effective therapy for CLL and that low-dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression. © 2014 Wiley Periodicals, Inc.

  2. Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

    Science.gov (United States)

    Hubert, Astrid; Bochenek, Magdalena L; Schütz, Eva; Gogiraju, Rajinikanth; Münzel, Thomas; Schäfer, Katrin

    2017-09-01

    Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the

  3. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    2015-11-01

    Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

  4. Deletion of Core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development

    Science.gov (United States)

    Wu, Mengrui; Li, Chenguan; Zhu, Guochun; Wang, Yiping; Jules, Joel; Lu, Yun; McConnell, Matthew; Wang, Yong-Jun; Shao, Jian-Zhong; Li, Yi-Ping; Chen, Wei

    2015-01-01

    Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage causing alveolus defects which led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wildtype mice indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors impacted bone

  5. The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

    Czech Academy of Sciences Publication Activity Database

    Dmytrenko, Lesia; Cicanič, Michal; Anděrová, Miroslava; Voříšek, Ivan; Ottersen, O. P.; Syková, Eva; Vargová, Lýdia

    2013-01-01

    Roč. 8, č. 7 (2013), e68044 E-ISSN 1932-6203 R&D Projects: GA ČR GA309/09/1597; GA ČR(CZ) GAP303/11/2378; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : extracellular space * diffusion parameters * cell swelling Subject RIV: FH - Neurology Impact factor: 3.534, year: 2013

  6. Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice.

    Science.gov (United States)

    Zou, Junhuang; Zheng, Tihua; Ren, Chongyu; Askew, Charles; Liu, Xiao-Ping; Pan, Bifeng; Holt, Jeffrey R; Wang, Yong; Yang, Jun

    2014-05-01

    Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

  7. Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages.

    Science.gov (United States)

    Han, Kyudong; Sen, Shurjo K; Wang, Jianxin; Callinan, Pauline A; Lee, Jungnam; Cordaux, Richard; Liang, Ping; Batzer, Mark A

    2005-01-01

    Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of approximately 18 kb of sequence from the human genome and approximately 15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.

  8. Bilateral granulosa cell tumors: a novel malignant manifestation of multiple endocrine neoplasia 1 syndrome found in a patient with a rare menin in-frame deletion

    Directory of Open Access Journals (Sweden)

    Hall MJ

    2015-02-01

    Full Text Available Michael J Hall,1 Julie Innocent,2 Christina Rybak,1 Colleen Veloski,3 Walter J Scott,4 Hong Wu,5 John A Ridge,4 John P Hoffman,4 Hossein Borghaei,2 Aruna Turaka,6 Mary B Daly1 1Department of Clinical Genetics, 2Department of Medical Oncology, 3Department of Internal Medicine, 4Department of Surgical Oncology, 5Department of Pathology, 6Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA Introduction: Multiple endocrine neoplasia 1 (MEN1 is a cancer syndrome resulting from mutations of the MEN1 gene. The syndrome is characterized by neoplasia of the parathyroid and pituitary glands, and malignant tumors of the endocrine pancreas. Other manifestations include benign lipomas, angiofibromas, and carcinoid tumors commonly originating in the colon, thymus, and lung. This is the first report of MEN1 syndrome manifesting as bilateral granulosa cell ovarian tumors, and which is associated with a rare intronic mutation of the MEN1 gene. Case report: A 41-year-old woman presented with abdominal pain, increasing abdominal girth, and dysmenorrhea. Ultrasound demonstrated enlarged ovaries and uterine fibroids. After an exploratory laparotomy, she subsequently underwent bilateral salpingo–oophorectomy with hysterectomy where the pathology revealed bilateral cystic granulosa cell tumors of the ovaries. Additional workup including computed tomography imaging discovered a thymic mass, which the pathology showed was malignant, along with a pancreatic mass suspicious for a neuroendocrine tumor. Hyperparathyroidism was also discovered and was found to be secondary to a parathyroid adenoma. Genetic testing revealed an exceedingly rare mutation in the MEN1 gene (c.654 + 1 G>A. Discussion: Mutations of the menin gene leading to MEN1 syndrome are classically nonsense or missense mutations producing a dysfunctional protein product. Recently, researchers described a novel mutation of MEN1 (c.654 + 1 G>A in a male proband meeting the criteria

  9. Heterozygous deletion at the RLN1 locus in a family with testicular germ cell cancer identified by integrating copy number variation data with phenome and interactome information

    DEFF Research Database (Denmark)

    Edsgärd, D; Scheel, M; Hansen, N T

    2011-01-01

    -associated genes among loci targeted by CNVs. The top-ranked candidate, RLN1, encoding a Relaxin-H1 peptide, although only detected in one of the families, was selected for further investigations. Validation of the CNV at the RLN1 locus was performed as an association study using qPCR with 106 sporadic testicular...... GCT patients and 200 healthy controls. Observed CNV frequencies of 1.9% among cases and 1.5% amongst controls were not significantly different and this was further confirmed by CNV data extracted from a genome-wide analysis of 189 cases and 380 controls, where similar frequencies of 2.2% were observed....... Collectively, the findings show that a heterozygous loss at the RLN1 locus is not a genetic factor mediating high population-wide risk for testicular germ cell tumour, but do not exclude a contribution of this aberration in some cases of cancer. The preliminary expression data suggest a possible role...

  10. A Rare Syndrome of Deletion in 2 Siblings

    Directory of Open Access Journals (Sweden)

    Aravindhan Veerapandiyan MBBS

    2017-08-01

    Full Text Available The Glutamate receptor, ionotropic, delta 2 gene codes for an ionotropic glutamate delta-2 receptor, which is selectively expressed in cerebellar Purkinje cells, and facilitates cerebellar synapse organization and transmission. The phenotype associated with the deletion of Glutamate receptor, ionotropic, delta 2 gene in humans was initially defined in 2013. In this case report, the authors describe 2 brothers who presented with developmental delay, tonic upward gaze, nystagmus, oculomotor apraxia, hypotonia, hyperreflexia, and ataxia. They were found to have a homozygous intragenic deletion within the Glutamate receptor, ionotropic, delta 2 gene at exon 2. Our patients serve as an addition to the literature of previously reported children with this rare clinical syndrome associated with Glutamate receptor, ionotropic, delta 2 deletion.

  11. Heterozygous deletion at the RLN1 locus in a family with testicular germ cell cancer identified by integrating copy number variation data with phenome and interactome information

    DEFF Research Database (Denmark)

    Edsgard, Stefan Daniel; Scheel, M.; Hansen, Niclas Tue

    2011-01-01

    ‐associated genes among loci targeted by CNVs. The top‐ranked candidate, RLN1, encoding a Relaxin‐H1 peptide, although only detected in one of the families, was selected for further investigations. Validation of the CNV at the RLN1 locus was performed as an association study using qPCR with 106 sporadic testicular...... GCT patients and 200 healthy controls. Observed CNV frequencies of 1.9% among cases and 1.5% amongst controls were not significantly different and this was further confirmed by CNV data extracted from a genome‐wide analysis of 189 cases and 380 controls, where similar frequencies of 2.2% were observed...... and spermatids. Collectively, the findings show that a heterozygous loss at the RLN1 locus is not a genetic factor mediating high population‐wide risk for testicular germ cell tumour, but do not exclude a contribution of this aberration in some cases of cancer. The preliminary expression data suggest a possible...

  12. Mice deleted for cell division cycle 73 gene develop parathyroid and uterine tumours: model for the hyperparathyroidism-jaw tumour syndrome.

    Science.gov (United States)

    Walls, G V; Stevenson, M; Lines, K E; Newey, P J; Reed, A A C; Bowl, M R; Jeyabalan, J; Harding, B; Bradley, K J; Manek, S; Chen, J; Wang, P; Williams, B O; Teh, B T; Thakker, R V

    2017-07-13

    The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by occurrence of parathyroid tumours, often atypical adenomas and carcinomas, ossifying jaw fibromas, renal tumours and uterine benign and malignant neoplasms. HPT-JT is caused by mutations of the cell division cycle 73 (CDC73) gene, located on chromosome 1q31.2 and encodes a 531 amino acid protein, parafibromin. To facilitate in vivo studies of Cdc73 in tumourigenesis we generated conventional (Cdc73 +/- ) and conditional parathyroid-specific (Cdc73 +/L /PTH-Cre and Cdc73 L/L /PTH-Cre) mouse models. Mice were aged to 18-21 months and studied for survival, tumour development and proliferation, and serum biochemistry, and compared to age-matched wild-type (Cdc73 +/+ and Cdc73 +/+ /PTH-Cre) littermates. Survival of Cdc73 +/- mice, when compared to Cdc73 +/+ mice was reduced (Cdc73 +/- =80%; Cdc73 +/+ =90% at 18 months of age, Pfourfold higher than that in parathyroid glands of wild-type littermates (P<0.0001). Cdc73 +/- , Cdc73 +/L /PTH-Cre and Cdc73 L/L /PTH-Cre mice had higher mean serum calcium concentrations than wild-type littermates, and Cdc73 +/- mice also had increased mean serum parathyroid hormone (PTH) concentrations. Parathyroid tumour development, and elevations in serum calcium and PTH, were similar in males and females. Cdc73 +/- mice did not develop bone or renal tumours but female Cdc73 +/- mice, at 18 months of age, had uterine neoplasms comprising squamous metaplasia, adenofibroma and adenomyoma. Uterine neoplasms, myometria and jaw bones of Cdc73 +/- mice had increased proliferation rates that were 2-fold higher than in Cdc73 +/+ mice (P<0.05). Thus, our studies, which have established mouse models for parathyroid tumours and uterine neoplasms that develop in the HPT-JT syndrome, provide in vivo models for future studies of these tumours.

  13. Concurrent deletion of 16q23 and PTEN is an independent prognostic feature in prostate cancer.

    Science.gov (United States)

    Kluth, Martina; Runte, Frederic; Barow, Philipp; Omari, Jazan; Abdelaziz, Zaid M; Paustian, Lisa; Steurer, Stefan; Christina Tsourlakis, Maria; Fisch, Margit; Graefen, Markus; Tennstedt, Pierre; Huland, Hartwig; Michl, Uwe; Minner, Sarah; Sauter, Guido; Simon, Ronald; Adam, Meike; Schlomm, Thorsten

    2015-11-15

    The deletion of 16q23-q24 belongs to the most frequent chromosomal changes in prostate cancer, but the clinical consequences of this alteration have not been studied in detail. We performed fluorescence in situ hybridization analysis using a 16q23 probe in more than 7,400 prostate cancers with clinical follow-up data assembled in a tissue microarray format. Chromosome 16q deletion was found in 21% of cancers, and was linked to advanced tumor stage, high Gleason grade, accelerated cell proliferation, the presence of lymph node metastases (p Deletion was more frequent in ERG fusion-positive (27%) as compared to ERG fusion-negative cancers (16%, p deletions including phosphatase and tensin homolog (PTEN) (p deletion of 16q was linked to early biochemical recurrence independently from the ERG status (p deletion of 16q alone. Multivariate modeling revealed that the prognostic value of 16q/PTEN deletion patterns was independent from the established prognostic factors. In summary, the results of our study demonstrate that the deletion of 16q and PTEN cooperatively drives prostate cancer progression, and suggests that deletion analysis of 16q and PTEN could be of important clinical value particularly for preoperative risk assessment of the clinically most challenging group of low- and intermediated grade prostate cancers. © 2015 UICC.

  14. Cell junction protein armadillo repeat gene deleted in velo-cardio-facial syndrome is expressed in the skin and colocalizes with autoantibodies of patients affected by a new variant of endemic pemphigus foliaceus in Colombia.

    Science.gov (United States)

    Abreu-Velez, Ana Maria; Yi, Hong; Howard, Michael S

    2017-10-01

    We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu). El Bagre-EPF differs from other types of EPF clinically, epidemiologically, immunologically and in its target antigens. We reported the presence of patient autoantibodies colocalizing with armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), a catenin cell junction protein colocalizing with El Bagre-EPF autoantibodies in the heart and within pilosebaceous units along their neurovascular supply routes. Here we investigate the presence of ARVCF in skin and its possibility as a cutaneous El Bagre-EPF antigen. We used a case-control study, testing sera of 45 patients and 45 controls via direct and indirect immunofluorescence (DIF/IIF), confocal microscopy, immunoelectron microscopy and immunoblotting for the presence of ARVCF and its relationship with El Bagre-EPF autoantibodies in the skin. We also immunoadsorbed samples with desmoglein 1 (Dsg1) ectodomain (El Bagre-EPF antigen) by incubating with the positive ARVCF samples from DIF and IIF. ARVCF was expressed in all the samples from the cases and controls. Immunoadsorption with Dsg1 on positive ARVCF immunofluorescence DIF/IIF cases showed that the immune response was present against non-desmoglein 1 antigen(s). Overall, 40/45 patients showed colocalization of their autoantibodies with ARVCF in the epidermis; no controls from the endemic area displayed colocalization. We demonstrate that ARVCF is expressed in many areas of human skin, and colocalizes with the majority of El Bagre-EPF autoantibodies as a putative antigen.

  15. Deletion mutants of region E1 a of AD12 E1 plasmids: Effect on oncogenic transformation

    NARCIS (Netherlands)

    Bos, J.L.; Jochemsen, A.G.; Bernards, R.A.; Schrier, P.I.; Ormondt, H. van; Eb, A.J. van der

    1983-01-01

    Plasmids containing the El region of Ad12 DNA can transform certain rodent cells into oncogenic cells. To study the role of the Ela subregion in the process of oncogenic transformation, Ad12 region El mutants carrying deletions in the Ela region were constructed. Deletion mutants pR7 and pR8 affect

  16. Prospective Study of a Cohort of Russian Nijmegen Breakage Syndrome Patients Demonstrating Predictive Value of Low Kappa-Deleting Recombination Excision Circle (KREC Numbers and Beneficial Effect of Hematopoietic Stem Cell Transplantation (HSCT

    Directory of Open Access Journals (Sweden)

    Elena Deripapa

    2017-07-01

    Full Text Available BackgroundNijmegen breakage syndrome (NBS is a combined primary immunodeficiency with DNA repair defect, microcephaly, and other phenotypical features. It predominantly occurs in Slavic populations that have a high frequency of carriers with the causative NBN gene c.657_661del5 mutation. Due to the rarity of the disease in the rest of the world, studies of NBS patients are few. Here, we report a prospective study of a cohort of Russian NBS patients.Methods35 Russian NBS patients of ages 1–19 years, referred to our Center between years 2012 and 2016, were prospectively studied.ResultsDespite the fact that in 80% of the patients microcephaly was diagnosed at birth or shortly thereafter, the average delay of NBS diagnosis was 6.5 years. Though 80% of the patients had laboratory signs of immunodeficiency, only 51% of the patients experienced significant infections. Autoimmune complications including interstitial lymphocytic lung disease and skin granulomas were noted in 34%, malignancies—in 57% of the patients. T-cell excision circle (TREC/kappa-deleting recombination excision circle (KREC levels were low in the majority of patients studied. Lower KREC levels correlated with autoimmune and oncological complications. Fifteen patients underwent hematopoietic stem cell transplantation (HSCT, 10 of them were alive and well, with good graft function. Three patients in the HSCT group and five non-transplanted patients died; tumor progression being the main cause of death. The probability of the overall survival since NBS diagnosis was 0.76 in the HSCT group and 0.3 in the non-transplanted group.ConclusionBased on our findings of low TRECs in most NBS patients, independent of their age, TREC detection can be potentially useful for detection of NBS patients during neonatal screening. KREC concentration can be used as a prognostic marker of disease severity. HSCT is a viable treatment option in NBS and should be especially considered in patients with

  17. Heme oxygenase-1 deletion affects stress erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Yu-An Cao

    Full Text Available Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1 deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/- or hmox(+/+ bone marrow cells, we evaluated (i the erythrocyte parameters in the peripheral blood; (ii the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii the patterns of histological iron staining; and (iv the number of Mac-1(+-cells expressing TNF-α. In the spleens of mice that received hmox(+/- cells, we show (i decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii increases in the insoluble iron levels and decreases in the soluble iron levels; (iii increased numbers of Mac-1(+-cells expressing TNF-α; and (iv decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.

  18. Effects of the deletion of early region 4 (E4 open reading frame 1 (orf1, orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

    Directory of Open Access Journals (Sweden)

    Michael A Thomas

    Full Text Available The global health burden engendered by human immunodeficiency virus (HIV-induced acquired immunodeficiency syndrome (AIDS is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1 through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and

  19. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

    Science.gov (United States)

    Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

    2013-01-01

    The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a

  20. Frequency of heterozygous TET2 deletions in myeloproliferative neoplasms

    Directory of Open Access Journals (Sweden)

    Joseph Tripodi

    2010-09-01

    Full Text Available Joseph Tripodi1, Ronald Hoffman1, Vesna Najfeld2, Rona Weinberg31The Myeloproliferative Disorders Program, Tisch Cancer Institute, Department of Medicine and 2Department of Medicine and Pathology, Mount Sinai School of Medicine, 3The Myeloproliferative Disorders Program, Cellular Therapy Laboratory, The New York Blood Center, New York, NY, USAAbstract: The Philadelphia chromosome (Ph-negative myeloproliferative neoplasms (MPNs, including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a group of clonal hematopoietic stem cell disorders with overlapping clinical and cytogenetic features and a variable tendency to evolve into acute leukemia. These diseases not only share overlapping chromosomal abnormalities but also a number of acquired somatic mutations. Recently, mutations in a putative tumor suppressor gene, ten-eleven translocation 2 (TET2 on chromosome 4q24 have been identified in 12% of patients with MPN. Additionally 4q24 chromosomal rearrangements in MPN, including TET2 deletions, have also been observed using conventional cytogenetics. The goal of this study was to investigate the frequency of genomic TET2 rearrangements in MPN using fluorescence in situ hybridization as a more sensitive method for screening and identifying genomic deletions. Among 146 MPN patients, we identified two patients (1.4% who showed a common 4q24 deletion, including TET2. Our observations also indicated that the frequency of TET2 deletion is increased in patients with an abnormal karyotype (5%.Keywords: TET2, myeloproliferative neoplasms, fluorescence in situ hybridization, cytogenetics

  1. Alterations on peripheral B cell subsets following an acute uncomplicated clinical malaria infection in children

    Directory of Open Access Journals (Sweden)

    Ng'ang'a Zipporah W

    2008-11-01

    Full Text Available Abstract Background The effects of Plasmodium falciparum on B-cell homeostasis have not been well characterized. This study investigated whether an episode of acute malaria in young children results in changes in the peripheral B cell phenotype. Methods Using flow-cytofluorimetric analysis, the B cell phenotypes found in the peripheral blood of children aged 2–5 years were characterized during an episode of acute uncomplicated clinical malaria and four weeks post-recovery and in healthy age-matched controls. Results There was a significant decrease in CD19+ B lymphocytes during acute malaria. Characterization of the CD19+ B cell subsets in the peripheral blood based on expression of IgD and CD38 revealed a significant decrease in the numbers of naive 1 CD38-IgD+ B cells while there was an increase in CD38+IgD- memory 3 B cells during acute malaria. Further analysis of the peripheral B cell phenotype also identified an expansion of transitional CD10+CD19+ B cells in children following an episode of acute malaria with up to 25% of total CD19+ B cell pool residing in this subset. Conclusion Children experiencing an episode of acute uncomplicated clinical malaria experienced profound disturbances in B cell homeostasis.

  2. Probabilistic cloning and deleting of quantum states

    International Nuclear Information System (INIS)

    Feng Yuan; Zhang Shengyu; Ying Mingsheng

    2002-01-01

    We construct a probabilistic cloning and deleting machine which, taking several copies of an input quantum state, can output a linear superposition of multiple cloning and deleting states. Since the machine can perform cloning and deleting in a single unitary evolution, the probabilistic cloning and other cloning machines proposed in the previous literature can be thought of as special cases of our machine. A sufficient and necessary condition for successful cloning and deleting is presented, and it requires that the copies of an arbitrarily presumed number of the input states are linearly independent. This simply generalizes some results for cloning. We also derive an upper bound for the success probability of the cloning and deleting machine

  3. An Fgf8 Mouse Mutant Phenocopies Human 22q11 Deletion Syndrome

    OpenAIRE

    Frank, Deborah U.; Fotheringham, Lori K.; Brewer, Judson A.; Muglia, Louis J.; Tristani-Firouzi, Martin; Capecchi, Mario R.; Moon, Anne M.

    2002-01-01

    Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormaliti...

  4. Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer.

    Science.gov (United States)

    Huang, S F; Xiao, S; Renshaw, A A; Loughlin, K R; Hudson, T J; Fletcher, J A

    1996-11-01

    Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer. In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and

  5. Molecular evidence for the induction of large interstitial deletions on mouse chromosome 8 by ionizing radiation

    International Nuclear Information System (INIS)

    Turker, Mitchell S.; Pieretti, Maura; Kumar, Sudha

    1997-01-01

    The P19H22 mouse embryonal carcinoma cell line is characterized by a hemizygous deficiency for the chromosome 8 encoded aprt (adenine phosphoribosyltransferase) gene and heterozygosity for many chromosome 8 loci. We have previously demonstrated that this cell line is suitable for mutational studies because it is permissive of events ranging in size from base-pair substitutions at the aprt locus to apparent loss of chromosome 8. Large mutational events, defined by loss of the remaining aprt allele, were found to predominate in spontaneous mutants and those induced by ionizing radiation. In this study we have used a PCR based assay to screen for loss of heterozygosity at microsatellite loci both proximal and distal to aprt in 137 Cs-induced and spontaneous aprt mutants. This approach allowed us to distinguish apparent interstitial deletional events from apparent recombinational events. Significantly, 32.5% (26 of 80) of the mutational events induced by 137 Cs appeared to be interstitial deletions as compared with 7.7% (6 of 78) in the spontaneous group. This difference was statistically significant (p 137 Cs caused a significant number of deletion mutations. Most 137 Cs-induced interstitial deletions were larger than 6 cM, whereas none of the spontaneous deletions were larger than 6 cM. These results provide further support for the notion that ionizing radiation induces deletion mutations and validate the use of the P19H22 cell line for the study of events induced by ionizing radiation

  6. Association of BIM Deletion Polymorphism and BIM-γ RNA Expression in NSCLC with EGFR Mutation.

    Science.gov (United States)

    Isobe, Kazutoshi; Kakimoto, Atsushi; Mikami, Tetsuo; Kaburaki, Kyohei; Kobayashi, Hiroshi; Yoshizawa, Takahiro; Makino, Takashi; Otsuka, Hajime; Sano, G O; Sugino, Keishi; Sakamoto, Susumu; Takai, Yujiro; Tochigi, Naobumi; Iyoda, Akira; Homma, Sakae

    This pilot study assessed the association of BIM deletion polymorphism and BIM RNA isoform in patients with EGFR-positive non-small cell lung cancer (NSCLC). The study included 33 patients with EGFR-positive NSCLC treated with gefitinib. BIM deletion polymorphism and BIM RNA isoform (EL/L/S/γ) were determined by polymerase chain reaction (PCR). BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism inside tumors (p=0.038) and around tumors (p=0.0024). Relative BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism (p=0.0017). Patients with BIM-γ had significantly shorter progression-free survival than those without BIM-γ (median: 304 vs. 732 days; p=0.023). Expression of BIM-γ mRNA and BIM deletion polymorphism were strongly associated. BIM-γ overexpression may have a role in apoptosis related to EGFR-tyrosine kinase inhibitor. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  7. Exploration of methods to localize DNA sequences missing from c-locus deletions

    International Nuclear Information System (INIS)

    Albritton, L.M.; Russell, L.B.; Montgomery, C.S.

    1987-01-01

    The authors have earlier characterized a large number of radiation-induced mutations at the c locus (on Chromosome 7) through genetic analysis, including extensive complementation tests. Based on this work, they have postulated that many of these mutations are deletions of various lengths, overlapping at c (the marker used in the mutation-rate experiments that generated the mutants). It was possible to apportion these deletions among 13 complementation groups and to fit them to a linear map of 8 functional units. Collectively, the deletions extend from a point between tp and c to one between sh-1 and Hbb, i.e., a genetic distance of from 6 to 10 cM, corresponding to at least 10 4 Kb of DNA. This year, the authors completed a pilot study designed to explore methods for finding DNA sequences that map to the region covered by the various c-deletions. The general plan was to probe DNA with clones derived from Chromosome-7-enriched libraries or with sequences known (or suspected) to reside in Chromosome 7. Three methods were explored for deriving the c-region-deficient DNA: (a) from mouse-hamster somatic-cell hydrids retaining a deleted mouse Chromosome 7, but no homologue; (b) from F 1 hybrids of M. musculus domesticus (carrying a c-locus deletion) by M. spretus; and (c) from F 1 hybrids of M. domesticus stocks carrying complementing deletions

  8. Budget Impact Analysis of Afatinib for First-Line Treatment of Patients with Metastatic Non-Small Cell Lung Cancer with Epidermal Growth Factor Receptor Exon 19 Deletions or Exon 21 Substitution Mutations in a U.S. Health Plan.

    Science.gov (United States)

    Graham, Jonathan; Earnshaw, Stephanie; Burslem, Kate; Lim, Jonathan

    2018-06-01

    Afatinib is 1 of 3 tyrosine kinase inhibitors approved in the United States for the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions (del19) or exon 21 (L858R) substitution mutations. In clinical trials, afatinib has demonstrated improvement in progression-free survival versus standard chemotherapy and gefitinib. To analyze the impact of increases in afatinib treatment share on the cost and health outcomes in a commercial health plan in the United States. A decision model was developed to evaluate the budget impact of increases in afatinib share for the first-line treatment of patients with metastatic NSCLC with EGFR del19 or L858R substitution mutations over a 5-year time horizon. The model compared the total annual costs for a health plan with 1 million covered lives in a scenario in which afatinib share increased 5 percentage points annually to one in which all treatment shares remained constant over time. The number of patients eligible for treatment was estimated using published incidence data. Therapies included in the model were afatinib, erlotinib, gefitinib, and the chemotherapy doublet, pemetrexed in combination with cisplatin. The mean time spent by patients in progression-free and progressive disease states was based on survival data from clinical trials and a network meta-analysis. Therapy-related costs included monthly drug acquisition and administration costs and costs of managing adverse reactions. Disease management costs were also assessed in the model. Scenario analyses were performed to assess alternative scenarios of afatinib treatment share. Additionally, a one-way sensitivity analysis was performed to test the robustness of the model, given parameter uncertainty. Using the base-case parameter assumptions and a 5-percentage-point annual increase in afatinib treatment share, we estimated the total budget increases in years 1 through 5

  9. Interallelic class switch recombination contributes significantly to class switching in mouse B cells.

    Science.gov (United States)

    Reynaud, Stéphane; Delpy, Laurent; Fleury, Laurence; Dougier, Hei-Lanne; Sirac, Christophe; Cogné, Michel

    2005-05-15

    Except for the expression of IgM and IgD, DNA recombination is constantly needed for the expression of other Ig classes and subclasses. The predominant path of class switch recombination (CSR) is intrachromosomal, and the looping-out and deletion model has been abundantly documented. However, switch regions also occasionally constitute convenient substrates for interchromosomal recombination, since it is noticeably the case in a number of chromosomal translocations causing oncogene deregulation in the course of lymphoma and myeloma. Although asymmetric accessibility of Ig alleles should theoretically limit its occurrence, interallelic CSR was shown to occur at low levels during IgA switching in rabbit, where the definition of allotypes within both V and C regions helped identify interchromosomally derived Ig. Thus, we wished to evaluate precisely interallelic CSR frequency in mouse B cells, by using a system in which only one allele (of b allotype) could express a functional VDJ region, whereas only interallelic CSR could restore expression of an excluded (a allotype) allele. In our study, we show that interchromosomal recombination of V(H) and Cgamma or Calpha occurs in vivo in B cells at a frequency that makes a significant contribution to physiological class switching: trans-association of V(H) and C(H) genes accounted for 7% of all alpha mRNA, and this frequency was about twice higher for the gamma3 transcripts, despite the much shorter distance between the J(H) region and the Cgamma3 gene, thus confirming that this phenomenon corresponded to site-specific switching and not to random recombination between long homologous loci.

  10. Seven gene deletions in seven days

    DEFF Research Database (Denmark)

    Ingemann Jensen, Sheila; Lennen, Rebecca; Herrgard, Markus

    2015-01-01

    Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering ...

  11. A New Intergenic α-Globin Deletion (α-αΔ125) Found in a Kabyle Population.

    Science.gov (United States)

    Singh, Amrathlal Rabbind; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-01-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

  12. The mitochondrial DNA 4,977-bp deletion and its implication in copy number alteration in colorectal cancer

    Science.gov (United States)

    2011-01-01

    Background Qualitative and quantitative changes in human mitochondrial DNA (mtDNA) have been implicated in various cancer types. A 4,977 bp deletion in the major arch of the mitochondrial genome is one of the most common mutations associated with a variety of human diseases and aging. Methods We conducted a comprehensive study on clinical features and mtDNA of 104 colorectal cancer patients in the Wenzhou area of China. In particular, using a quantitative real time PCR method, we analyzed the 4,977 bp deletion and mtDNA content in tumor tissues and paired non-tumor areas from these patients. Results We found that the 4,977 bp deletion was more likely to be present in patients of younger age (≤65 years, p = 0.027). In patients with the 4,977 bp deletion, the deletion level decreased as the cancer stage advanced (p = 0.031). Moreover, mtDNA copy number in tumor tissues of patients with this deletion increased, both compared with that in adjacent non-tumor tissues and with in tumors of patients without the deletion. Such mtDNA content increase correlated with the levels of the 4,977 bp deletion and with cancer stage (p deletion may play a role in the early stage of colorectal cancer, and it is also implicated in alteration of mtDNA content in cancer cells. PMID:21232124

  13. Skin fibroblasts from a D-deletion type retinoblastoma patient are abnormally X-ray sensitive

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Nove, J.; Little, J.B.

    1977-01-01

    Retinoblastoma is a rare malignant eye tumour that appears either spontaneously or in genetically predisposed persons. The latter group is composed of persons who inherit the tumour with a dominant mode of transmission (the familial type) and those who have a deletion in the long arm of chromosome 13 referred to as the D-deletion type. When this deletion is present it is observed in many somatic cells and is often associated with structural defects. Survivors of the genetic forms of retinoblastoma have an increased risk of the development of cancers at other sites. A single genetic locus is unlikely to predispose many somatic cells to tumour formation unless a fundamental molecular defect, possibly related to DNA repair, is present. In order to investigate this hypothesis a study was made of the in vitro X-ray sensitivity of skin fibroblasts derived from three retinoblastoma patients, comprising a pair of twins with the familial type accompanied by no gross chromosome abnormalities, and a patient with the D-deletion type. It was found that fibroblasts derived from the D-deletion patient were significantly more radiosensitive than those from the other two patients. X-ray survival curves are shown. It is concluded that skin fibroblasts derived from a patient with the D-deletion variant of retinoblastoma are abnormally radiosensitive. Future investigations may indicate a specific defect in molecular repair of DNA that will explain the predisposition of these patients to the development of other tumours. (U.K.)

  14. 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.

  15. 46 CFR 67.171 - Deletion; requirement and procedure.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  16. 19 CFR 142.49 - Deletion of C-4 Code.

    Science.gov (United States)

    2010-04-01

    .... Entry filers may delete C-4 Codes from Line Release by notifying the port director in writing on a Deletion Data Loading Sheet. Such notification shall state the C-4 Code which is to be deleted, the port... TREASURY (CONTINUED) ENTRY PROCESS Line Release § 142.49 Deletion of C-4 Code. (a) By Customs. A port...

  17. A recombinant E1-deleted porcine adenovirus-3 as an expression vector

    International Nuclear Information System (INIS)

    Zakhartchouk, Alexander; Zhou Yan; Tikoo, Suresh Kumar

    2003-01-01

    Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B large coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B large of PAV-3 and also complemented PAV214 (E1A+E1B small deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B large coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B small + E1B large ) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B large was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines

  18. Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions

    NARCIS (Netherlands)

    Glogauer, Michael; Marchal, Christophe C.; Zhu, Fei; Worku, Aelaf; Clausen, Björn E.; Foerster, Irmgard; Marks, Peter; Downey, Gregory P.; Dinauer, Mary; Kwiatkowski, David J.

    2003-01-01

    Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To

  19. A nine-nucleotide deletion and splice variation in the coding region of the interferon induced ISG12 gene

    DEFF Research Database (Denmark)

    Smidt, Kamille; Hansen, Lise Lotte; Søgaard, T Max M

    2003-01-01

    distributed between ISG12 and ISG12-S in breast carcinoma cells, in cancer cell lines and in cervical cytobrush material with neoplastic lesions. In addition, we have found a nine-nucleotide deletion situated in exon 4 of the ISG12 gene. This deletion leads to a three-amino-acid deletion (AMA) in the putative...... ISG12 gene products, ISG12Δ and ISG12-SΔ. We have determined the prevalence of the deletion ISG12Δ in normal and neoplastic cells. Homozygosity ISG12(0/0) and ISG12(Δ/Δ), and heterozygosity ISG12(0/Δ) were found, although the ISG12(Δ/Δ) genotype was rare. In heterozygous cells from cytobrush material...

  20. Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.

    Science.gov (United States)

    Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on t...

  1. Kcne2 deletion impairs insulin secretion and causes type 2 diabetes mellitus.

    Science.gov (United States)

    Lee, Soo Min; Baik, Jasmine; Nguyen, Dara; Nguyen, Victoria; Liu, Shiwei; Hu, Zhaoyang; Abbott, Geoffrey W

    2017-06-01

    Type 2 diabetes mellitus (T2DM) represents a rapidly increasing threat to global public health. T2DM arises largely from obesity, poor diet, and lack of exercise, but it also involves genetic predisposition. Here we report that the KCNE2 potassium channel transmembrane regulatory subunit is expressed in human and mouse pancreatic β cells. Kcne2 deletion in mice impaired glucose tolerance as early as 5 wk of age in pups fed a Western diet, ultimately causing diabetes. In adult mice fed normal chow, skeletal muscle expression of insulin receptor β and insulin receptor substrate 1 were down-regulated 2-fold by Kcne2 deletion, characteristic of T2DM. Kcne2 deletion also caused extensive pancreatic transcriptome changes consistent with facets of T2DM, including endoplasmic reticulum stress, inflammation, and hyperproliferation. Kcne2 deletion impaired β-cell insulin secretion in vitro up to 8-fold and diminished β-cell peak outward K + current at positive membrane potentials, but also left-shifted its voltage dependence and slowed inactivation. Interestingly, we also observed an aging-dependent reduction in β-cell outward currents in both Kcne2 +/+ and Kcne2 - / - mice. Our results demonstrate that KCNE2 is required for normal β-cell electrical activity and insulin secretion, and that Kcne2 deletion causes T2DM. KCNE2 may regulate multiple K + channels in β cells, including the T2DM-linked KCNQ1 potassium channel α subunit.-Lee, S. M., Baik, J., Nguyen, D., Nguyen, V., Liu, S., Hu, Z., Abbott, G. W. Kcne2 deletion impairs insulin secretion and causes type 2 diabetes mellitus. © FASEB.

  2. Deletion of Fanca or Fancd2 dysregulates Treg in mice

    Science.gov (United States)

    Du, Wei; Erden, Ozlem; Wilson, Andrew; Sipple, Jared M.; Schick, Jonathan; Mehta, Parinda; Myers, Kasiani C.; Steinbrecher, Kris A.; Davies, Stella M.

    2014-01-01

    Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca−/− or Fancd2−/− BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca−/− or Fancd2−/− mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25+Foxp3+ Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25+Foxp3+ Tregs of Fanca−/− or Fancd2−/− mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients. PMID:24501220

  3. Deletion of Fanca or Fancd2 dysregulates Treg in mice.

    Science.gov (United States)

    Du, Wei; Erden, Ozlem; Wilson, Andrew; Sipple, Jared M; Schick, Jonathan; Mehta, Parinda; Myers, Kasiani C; Steinbrecher, Kris A; Davies, Stella M; Pang, Qishen

    2014-03-20

    Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca(-/-) or Fancd2(-/-) BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca(-/-) or Fancd2(-/-) mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25(+)Foxp3(+) Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25(+)Foxp3(+) Tregs of Fanca(-/-) or Fancd2(-/-) mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients.

  4. Generating Bona Fide Mammalian Prions with Internal Deletions.

    Science.gov (United States)

    Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Chapuis, Jérôme; Ciric, Danica; Igel-Egalon, Angelique; Laude, Hubert; Béringue, Vincent; Rezaei, Human; Dron, Michel

    2016-08-01

    Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative

  5. Genomic clones of bovine parvovirus: Construction and effect of deletions and terminal sequence inversions on infectivity

    International Nuclear Information System (INIS)

    Shull, B.C.; Chen, K.C.; Lederman, M.; Stout, E.R.; Bates, R.C.

    1988-01-01

    Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytophathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Expression of capsid proteins from tranfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [ 35 S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus

  6. [Changes of biological behavioral of E. coli K1 after ppk1 gene deletion].

    Science.gov (United States)

    Peng, Liang; Pan, Jiayun; Luo, Su; Yang, Zhenghui; Huang, Mufang; Cao, Hong

    2014-06-01

    To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

  7. Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

    Science.gov (United States)

    Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

    2012-01-01

    The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

  8. Histone Deacetylase 3 Inhibition Overcomes BIM Deletion Polymorphism-Mediated Osimertinib Resistance in EGFR-Mutant Lung Cancer.

    Science.gov (United States)

    Tanimoto, Azusa; Takeuchi, Shinji; Arai, Sachiko; Fukuda, Koji; Yamada, Tadaaki; Roca, Xavier; Ong, S Tiong; Yano, Seiji

    2017-06-15

    Purpose: The BIM deletion polymorphism is associated with apoptosis resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, in non-small cell lung cancer (NSCLC) harboring EGFR mutations. Here, we investigated whether the BIM deletion polymorphism contributes to resistance against osimertinib, a third-generation EGFR-TKI. In addition, we determined the efficacy of a histone deacetylase (HDAC) inhibitor, vorinostat, against this form of resistance and elucidated the underlying mechanism. Experimental Design: We used EGFR -mutated NSCLC cell lines, which were either heterozygous or homozygous for the BIM deletion polymorphism, to evaluate the effect of osimertinib in vitro and in vivo Protein expression was examined by Western blotting. Alternative splicing of BIM mRNA was analyzed by RT-PCR. Results: EGFR -mutated NSCLC cell lines with the BIM deletion polymorphism exhibited apoptosis resistance to osimertinib in a polymorphism dosage-dependent manner, and this resistance was overcome by combined use with vorinostat. Experiments with homozygous BIM deletion-positive cells revealed that vorinostat affected the alternative splicing of BIM mRNA in the deletion allele, increased the expression of active BIM protein, and thereby induced apoptosis in osimertinib-treated cells. These effects were mediated predominantly by HDAC3 inhibition. In xenograft models, combined use of vorinostat with osimertinib could regress tumors in EGFR -mutated NSCLC cells homozygous for the BIM deletion polymorphism. Moreover, this combination could induce apoptosis even when tumor cells acquired EGFR -T790M mutations. Conclusions: These findings indicate the importance of developing HDAC3-selective inhibitors, and their combined use with osimertinib, for treating EGFR -mutated lung cancers carrying the BIM deletion polymorphism. Clin Cancer Res; 23(12); 3139-49. ©2016 AACR . ©2016 American Association for Cancer Research.

  9. Deletion 22q13.3 syndrome

    Directory of Open Access Journals (Sweden)

    Phelan Mary C

    2008-05-01

    Full Text Available Abstract The deletion 22q13.3 syndrome (deletion 22q13 syndrome or Phelan-McDermid syndrome is a chromosome microdeletion syndrome characterized by neonatal hypotonia, global developmental delay, normal to accelerated growth, absent to severely delayed speech, and minor dysmorphic features. The deletion occurs with equal frequency in males and females and has been reported in mosaic and non-mosaic forms. Due to lack of clinical recognition and often insufficient laboratory testing, the syndrome is under-diagnosed and its true incidence remains unknown. Common physical traits include long eye lashes, large or unusual ears, relatively large hands, dysplastic toenails, full brow, dolicocephaly, full cheeks, bulbous nose, and pointed chin. Behavior is autistic-like with decreased perception of pain and habitual chewing or mouthing. The loss of 22q13.3 can result from simple deletion, translocation, ring chromosome formation and less common structural changes affecting the long arm of chromosome 22, specifically the region containing the SHANK3 gene. The diagnosis of deletion 22q13 syndrome should be considered in all cases of hypotonia of unknown etiology and in individuals with absent speech. Although the deletion can sometimes be detected by high resolution chromosome analysis, fluorescence in situ hybridization (FISH or array comparative genomic hybridization (CGH is recommended for confirmation. Differential diagnosis includes syndromes associated with hypotonia, developmental delay, speech delay and/or autistic-like affect (Prader-Willi, Angelman, Williams, Smith-Magenis, Fragile X, Sotos, FG, trichorhinophalangeal and velocardiofacial syndromes, autism spectrum disorders, cerebral palsy. Genetic counseling is recommended and parental laboratory studies should be considered to identify cryptic rearrangements and detect parental mosaicism. Prenatal diagnosis should be offered for future pregnancies in those families with inherited rearrangements

  10. PTEN and DMBT1 homozygous deletion and expression in medulloblastomas and supratentorial primitive neuroectodermal tumors.

    Science.gov (United States)

    Inda, María Mar; Mercapide, Javier; Muñoz, Jorge; Coullin, Philippe; Danglot, Giséle; Tuñon, Teresa; Martínez-Peñuela, José María; Rivera, José María; Burgos, Juan J; Bernheim, Alain; Castresana, Javier S

    2004-12-01

    Medulloblastoma, which accounts for 20-25% of all childhood brain tumors, is defined as a primitive neuroectodermal tumor (PNET) located in the cerebellum. Supratentorial PNET are less frequent than medulloblastoma. But their clinical outcome is worse than in medulloblastomas. Chromosome 10q contains at least 2 tumor suppressor genes that might play a role in brain tumor development: PTEN and DMBT1. The aim of this study was to compare the status of homozygous deletion and expression of PTEN and DMBT1 genes in PNET primary tumor samples and cell lines. Homozygous deletions of PTEN and DMBT1 were studied in 32 paraffin-embedded PNET samples (23 medulloblastomas and 9 supratentorial PNET) and in 7 PNET cell lines, by differential PCR and by FISH. PTEN homozygous losses were demonstrated in 7 medulloblastomas (32%) and in no supratentorial PNET, while homozygous deletions of DMBT1 appeared in 1 supratentorial PNET (20%) and in 7 medulloblastomas (33%). No homozygous deletion of PTEN or DMBT1 was detected in any of the PNET cell lines either by differential PCR or by FISH. Expression study of the 2 genes was performed in the 7 PNET cell lines by RT-PCR. One PNET cell line lacked PTEN and DMBT1 expression, while 2 medulloblastoma cell lines did not express DMBT1. Our results add some positive data to the hypothesis that supratentorial PNETs and medulloblastomas might be genetically different.

  11. Some analogies between quantum cloning and quantum deleting

    International Nuclear Information System (INIS)

    Qiu Daowen

    2002-01-01

    We further verify the impossibility of deleting an arbitrary unknown quantum state, and also show it is impossible to delete two nonorthogonal quantum states as a consequence of unitarity of quantum mechanics. A quantum approximate (deterministic) deleting machine and a probabilistic (exact) deleting machine are constructed. The estimation for the global fidelity characterizing the efficiency of the quantum approximate deleting is given. We then demonstrate that unknown nonorthogonal states chosen from a set with their multiple copies can evolve into a linear superposition of multiple deletions and failure branches by a unitary process if and only if the states are linearly independent. It is notable that the proof for necessity is somewhat different from Pati's [Phys. Rev. Lett. 83, 2849 (1999)]. Another deleting machine for the input states that are unnecessarily linearly independent is also presented. The bounds on the success probabilities of these deleting machines are derived. So we expound some preliminary analogies between quantum cloning and deleting

  12. Familial deletion 18p syndrome: case report

    Directory of Open Access Journals (Sweden)

    Lemyre Emmanuelle

    2006-07-01

    Full Text Available Abstract Background Deletion 18p is a frequent deletion syndrome characterized by dysmorphic features, growth deficiencies, and mental retardation with a poorer verbal performance. Until now, five families have been described with limited clinical description. We report transmission of deletion 18p from a mother to her two daughters and review the previous cases. Case presentation The proband is 12 years old and has short stature, dysmorphic features and moderate mental retardation. Her sister is 9 years old and also has short stature and similar dysmorphic features. Her cognitive performance is within the borderline to mild mental retardation range. The mother also presents short stature. Psychological evaluation showed moderate mental retardation. Chromosome analysis from the sisters and their mother revealed the same chromosomal deletion: 46, XX, del(18(p11.2. Previous familial cases were consistent regarding the transmission of mental retardation. Our family differs in this regard with variable cognitive impairment and does not display poorer verbal than non-verbal abilities. An exclusive maternal transmission is observed throughout those families. Women with del(18p are fertile and seem to have a normal miscarriage rate. Conclusion Genetic counseling for these patients should take into account a greater range of cognitive outcome than previously reported.

  13. 78 FR 37525 - Procurement List; Deletions

    Science.gov (United States)

    2013-06-21

    .... Contracting Activity: Dept of the Air Force, FA7014 AFDW A7KI, Andrews AFB, MD. Service Type/Location: Laundry... Procurement List. SUMMARY: This action deletes products and services from the Procurement List that were... products and services listed below are no longer suitable for procurement by the Federal Government under...

  14. Sequence analysis of 17 NRXN1 deletions

    DEFF Research Database (Denmark)

    Hoeffding, Louise Kristine Enggaard; Hansen, Thomas; Ingason, Andrés

    2014-01-01

    into the molecular mechanisms governing such genomic rearrangements may increase our understanding of disease pathology and evolutionary processes. Here we analyse 17 carriers of non-recurrent deletions in the NRXN1 gene, which have been associated with neurodevelopmental disorders, e.g. schizophrenia, autism...

  15. Angiotensin Converting Enzyme Insertion/Deletion Gene ...

    African Journals Online (AJOL)

    Angiotensin Converting Enzyme Insertion/Deletion Gene Polymorphism: An Observational Study among Diabetic Hypertensive Subjects in Malaysia. ... Methods: The pharmacological effect of ACE inhibition on mean arterial pressure (MAP) and glomerular filtration rate (GFR) were observed among a total of 62 subjects for ...

  16. Obtaining a Proportional Allocation by Deleting Items

    NARCIS (Netherlands)

    Dorn, B.; de Haan, R.; Schlotter, I.; Röthe, J.

    2017-01-01

    We consider the following control problem on fair allocation of indivisible goods. Given a set I of items and a set of agents, each having strict linear preference over the items, we ask for a minimum subset of the items whose deletion guarantees the existence of a proportional allocation in the

  17. Union-Find with Constant Time Deletions

    DEFF Research Database (Denmark)

    Alstrup, Stephen; Thorup, Mikkel; Gørtz, Inge Li

    2014-01-01

    operations performed, and α_M/N_(n) is a functional inverse of Ackermann’s function. They left open the question whether delete operations can be implemented more efficiently than find operations, for example, in o(log n) worst-case time. We resolve this open problem by presenting a relatively simple...

  18. Mapping genomic deletions down to the base

    DEFF Research Database (Denmark)

    Dunø, Morten; Hove, Hanne; Kirchhoff, Maria

    2004-01-01

    the breakpoint of the third patient was mapped to a region previously predicted to be prone for rearrangements. One patient also harboured an inversion in connection with the deletion that disrupted the HDAC9 gene. All three patients showed clinical characteristics reminiscent of the hand-foot-genital syndrome...

  19. Deletions in the fifth alpha helix of HIV-1 matrix block virus release

    Energy Technology Data Exchange (ETDEWEB)

    Sanford, Bridget; Li, Yan; Maly, Connor J.; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Chen, Han [Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE (United States); Zhou, You [Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE (United States); Nebraska Center for Virology, Lincoln, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Nebraska Center for Virology, Lincoln, NE (United States)

    2014-11-15

    The matrix (MA) protein of HIV-1 is the N-terminal component of the Gag structural protein and is critical for the early and late stages of viral replication. MA contains five α-helices (α1–α5). Deletions in the N-terminus of α5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA∆96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA∆96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the α5 of MA inhibits virus release. - Highlights: • Deletions were identified in the C-terminus of matrix that block virus release. • These deletion mutants still multimerized and associated with membranes. • TEM showed the mutant particles were tethered to the cell surface. • Amino acid mutagenesis of the region did not affect release. • The data suggests that disruption of matrix structure blocks virus release.

  20. Deletions in the fifth alpha helix of HIV-1 matrix block virus release

    International Nuclear Information System (INIS)

    Sanford, Bridget; Li, Yan; Maly, Connor J.; Madson, Christian J.; Chen, Han; Zhou, You; Belshan, Michael

    2014-01-01

    The matrix (MA) protein of HIV-1 is the N-terminal component of the Gag structural protein and is critical for the early and late stages of viral replication. MA contains five α-helices (α1–α5). Deletions in the N-terminus of α5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA∆96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA∆96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the α5 of MA inhibits virus release. - Highlights: • Deletions were identified in the C-terminus of matrix that block virus release. • These deletion mutants still multimerized and associated with membranes. • TEM showed the mutant particles were tethered to the cell surface. • Amino acid mutagenesis of the region did not affect release. • The data suggests that disruption of matrix structure blocks virus release

  1. Delayed chromosomal instability caused by large deletion

    International Nuclear Information System (INIS)

    Ojima, M.; Suzuki, K.; Kodama, S.; Watanabe, M.

    2003-01-01

    Full text: There is accumulating evidence that genomic instability, manifested by the expression of delayed phenotypes, is induced by X-irradiation but not by ultraviolet (UV) light. It is well known that ionizing radiation, such as X-rays, induces DNA double strand breaks, but UV-light mainly causes base damage like pyrimidine dimers and (6-4) photoproducts. Although the mechanism of radiation-induced genomic instability has not been thoroughly explained, it is suggested that DNA double strand breaks contribute the induction of genomic instability. We examined here whether X-ray induced gene deletion at the hprt locus induces delayed instability in chromosome X. SV40-immortalized normal human fibroblasts, GM638, were irradiated with X-rays (3, 6 Gy), and the hprt mutants were isolated in the presence of 6-thioguanine (6-TG). A 2-fold and a 60-fold increase in mutation frequency were found by 3 Gy and 6 Gy irradiation, respectively. The molecular structure of the hprt mutations was determined by multiplex polymerase chain reaction of nine exons. Approximately 60% of 3 Gy mutants lost a part or the entire hprt gene, and the other mutants showed point mutations like spontaneous mutants. All 6 Gy mutants show total gene deletion. The chromosomes of the hprt mutants were analyzed by Whole Human Chromosome X Paint FISH or Xq telomere FISH. None of the point or partial gene deletion mutants showed aberrations of X-chromosome, however total gene deletion mutants induced translocations and dicentrics involving chromosome X. These results suggest that large deletion caused by DNA double strand breaks destabilizes chromosome structure, which may be involved in an induction of radiation-induced genomic instability

  2. T cell receptor-o deletion in human T cells

    NARCIS (Netherlands)

    M.C.M. Verschuren (Martie)

    1996-01-01

    textabstractThe immune system protects the body against pathogens such as bacteria, viruses, fungi, and parasites, when they pass the first line of body defence such as the skin or other epithelial and mucosal barriers. After penetration into the body, micro-organisms encounter the second line

  3. Velo-cardio-facial and partial DiGeorge phenotype in a child with interstitial deletion at 10p13 - implications for cytogenetics and molecular biology

    Energy Technology Data Exchange (ETDEWEB)

    Lipson, A.; Sholler, G.; Issacs, D. [Royal Alexandra Hospital for Children, Sydney (Australia)] [and others

    1996-11-11

    We report on a female with a interstitial deletion of 10p13 and a phenotype similar to that seen with the 22q deletion syndromes (DiGeorge/velo-cardio-facial). She had a posterior cleft palate, perimembranous ventricular septal defect, dyscoordinate swallowing, T-cell subset abnormalities, small ears, maxillary and mandibular hypoplasia, broad nasal bridge, deficient alae nasi, contractures of fingers and developmental delay. This could indicate homology of some developmental genes at 22q and 10p so that patients with the velocardiofacial phenotype who do not prove to be deleted on 22q are candidates for a 10p deletion. 58 refs., 3 figs.

  4. Genetics Home Reference: 17q12 deletion syndrome

    Science.gov (United States)

    ... with 17q12 deletion syndrome have delayed development (particularly speech and language delays), intellectual disability, or behavioral or psychiatric disorders. Behavioral and psychiatric conditions that have been reported in people with 17q12 deletion syndrome include autism ...

  5. Interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q

    International Nuclear Information System (INIS)

    Le Beau, M.M.; Epstein, N.D.; O'Brien, S.J.; Nienhuis, A.W.; Yang, Y.C.; Clark, S.C.; Rowley, J.D.

    1987-01-01

    The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, the authors localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint [del(5)(q13q33.3)], and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33[del(5)(q14q33.3)]. Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent from the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF, CSF-1, and FMS. The findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q- chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q)

  6. Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion

    Science.gov (United States)

    Vaszkó, Tibor; Papp, János; Krausz, Csilla; Casamonti, Elena; Géczi, Lajos; Olah, Edith

    2016-01-01

    Due to its palindromic setup, AZFc (Azoospermia Factor c) region of chromosome Y is one of the most unstable regions of the human genome. It contains eight gene families expressed mainly in the testes. Several types of rearrangement resulting in changes in the cumulative copy number of the gene families were reported to be associated with diseases such as male infertility and testicular germ cell tumors. The best studied AZFc rearrangement is gr/gr deletion. Its carriers show widespread phenotypic variation from azoospermia to normospermia. This phenomenon was initially attributed to different gr/gr subtypes that would eliminate distinct members of the affected gene families. However, studies conducted to confirm this hypothesis have brought controversial results, perhaps, in part, due to the shortcomings of the utilized subtyping methodology. This proof-of-concept paper is meant to introduce here a novel method aimed at subtyping AZFc rearrangements. It is able to differentiate the partial deletion and partial duplication subtypes of the Deleted in Azoospermia (DAZ) gene family. The keystone of the method is the determination of the copy number of the gene family member-specific variant(s) in a series of sequence family variant (SFV) positions. Most importantly, we present a novel approach for the correct interpretation of the variant copy number data to determine the copy number of the individual DAZ family members in the context of frequent interloci gene conversion.Besides DAZ1/DAZ2 and DAZ3/DAZ4 deletions, not yet described rearrangements such as DAZ2/DAZ4 deletion and three duplication subtypes were also found by the utilization of the novel approach. A striking feature is the extremely high concordance among the individual data pointing to a certain type of rearrangement. In addition to being able to identify DAZ deletion subtypes more reliably than the methods used previously, this approach is the first that can discriminate DAZ duplication subtypes as well

  7. Probabilistic deletion of copies of linearly independent quantum states

    International Nuclear Information System (INIS)

    Feng Jian; Gao Yunfeng; Wang Jisuo; Zhan Mingsheng

    2002-01-01

    We show that each of two copies of the nonorthogonal states randomly selected from a certain set S can be probabilistically deleted by a general unitary-reduction operation if and only if the states are linearly independent. We derive a tight bound on the best possible deleting efficiencies. These results for 2→1 probabilistic deleting are also generalized into the case of N→M deleting (N,M positive integers and N>M)

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  3. Genetics Home Reference: proximal 18q deletion syndrome

    Science.gov (United States)

    ... characteristic features. Most cases of proximal 18q deletion syndrome are the result of a new (de novo) deletion and are not inherited from a ... J, Fox PT, Stratton RF, Perry B, Hale DE. Recurrent interstitial deletions of proximal 18q: a new syndrome involving expressive speech delay. Am J Med Genet ...

  4. Frontonasal malformation with tetralogy of Fallot associated with a submicroscopic deletion of 22q11

    Energy Technology Data Exchange (ETDEWEB)

    Stratton, R.F. [South Texas Genetics Center, San Antonio, TX (United States); Payne, R.M. [Central Texas Genetics Center, Austin, TX (United States)

    1997-03-31

    We report on a 14-month-old girl with bifid nasal tip and tetralogy of Fallot. Several similar patients have been described with CNS or eye abnormalities. Chromosome analysis with FISH, using Oncor DiGeorge probes, confirmed a submicroscopic deletion of 22q11. Many patients with Shprintzen (velo-cardio-facial) syndrome have a similar deletion with conotruncal cardiac defects and an abnormal nasal shape, suggesting that a gene in this area, possibly affecting neural crest cells, influences facial and other midline development. 13 refs., 1 fig.

  5. Induction of DNA deletions after UV-light irradiation in yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Stepanova, A.N.; Koltovaya, N.A.

    2008-01-01

    We study mutagenic action of such a damaging agent as UV light, which can lead to DNA double-strand breaks (DSB). DNA deletions and gross rearrangements occur in process of DSB repair. We show that UV light induces deletion and rearrangement very efficiently. Analysis of efficacy of different types of repair shows that cell tries to repair DSBs with a combination of both homologous recombination (HR) and nonhomologous end joining (NHEJ) if available and that DSB repair by HR is more effective than by NHEJ in growing culture of haploid yeast

  6. An environment-mediated quantum deleter

    International Nuclear Information System (INIS)

    Srikanth, R.; Banerjee, Subhashish

    2007-01-01

    Environment-induced decoherence presents a great challenge to realizing a quantum computer. We point out the somewhat surprising fact that decoherence can be useful, indeed necessary, for practical quantum computation, in particular, for the effective erasure of quantum memory in order to initialize the state of the quantum computer. The essential point behind the deleter is that the environment, by means of a dissipative interaction, furnishes a contractive map towards a pure state. We present a specific example of an amplitude damping channel provided by a two-level system's interaction with its environment in the weak Born-Markov approximation. This is contrasted with a purely dephasing, non-dissipative channel provided by a two-level system's interaction with its environment by means of a quantum nondemolition interaction. We point out that currently used state preparation techniques, for example using optical pumping, essentially perform as quantum deleters

  7. Targeted deletion of hepatocyte Ikkβ confers growth advantages

    International Nuclear Information System (INIS)

    Koch, Katherine S.; Maeda, Shin; He, Guobin; Karin, Michael; Leffert, Hyam L.

    2009-01-01

    Mice lacking hepatocyte IKKβ (Ikkβ Δhep ) are defective in TNFα-activation of hepatocellular transcription factor NF-κB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkβ Δhep mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkβ Δhep hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G 0 → G 1 transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkβ also accelerates hepatocyte growth. Ikkβ Δhep hepatocyte proliferative responses show heightened sensitivity to TGFα and TNFα, and heightened expression of fibronectin, collagens I/III, nidogen, β-actin and integrin β1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkβ Δhep mice may also be caused by growth advantages of surviving Ikkβ-deleted hepatocytes.

  8. Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection.

    Science.gov (United States)

    Greninger, Alexander L; Pepper, Gregory; Shean, Ryan C; Cent, Anne; Palileo, Isabel; Kuypers, Jane M; Schiffer, Joshua T; Jerome, Keith R

    2017-01-01

    We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of four respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.

  9. Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

    DEFF Research Database (Denmark)

    Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

    2009-01-01

    With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

  10. Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

    International Nuclear Information System (INIS)

    Sparger, Ellen E.; Dubie, Robert A.; Shacklett, Barbara L.; Cole, Kelly S.; Chang, W.L.; Luciw, Paul A.

    2008-01-01

    Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-γ enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus

  11. Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions

    International Nuclear Information System (INIS)

    Urlaub, G.; Mitchell, P.J.; Kas, E.; Chasin, L.A.; Funanage, V.L.; Myoda, T.T.; Hamlin, J.

    1986-01-01

    A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of foreign DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells

  12. Deletion of a coordinate regulator of type 2 cytokine expression in mice

    Energy Technology Data Exchange (ETDEWEB)

    Mohrs, Markus; Blankespoor, Catherine M.; Wang, Zhi-En; Loots, Gaby G.; Hadeiba, Husein; Shinkai, Kanade; Rubin, Edward M.; Locksley, Richard M.

    2001-07-30

    Mechanisms underlying the differentiation of stable T helper subsets will be important in understanding how discrete types of immunity develop in response to different pathogens. An evolutionarily conserved {approx}400 base pair non-coding sequence in the IL-4/IL-13 intergenic region, designated CNS-1, was deleted in mice. The capacity to develop Th2 cells was compromised in vitro and in vivo in the absence of CNS-1. Despite the profound effect in T cells, mast cells from CNS-1-deleted mice maintained their capacity to produce IL-4. A T cell-specific element critical for optimal expression of type 2 cytokines may represent evolution of a regulatory sequence exploited by adaptive immunity.

  13. Bir1 Deletion Causes Malfunction of the Spindle Assembly Checkpoint and Apoptosis in Yeast

    International Nuclear Information System (INIS)

    Ren, Qun; Liou, Liang-Chun; Gao, Qiuqiang; Bao, Xiaoming; Zhang, Zhaojie

    2012-01-01

    Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H 2 O 2 treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

  14. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Muhammad Rauzan

    Full Text Available Chronic myeloid leukemia (CML treatment has been improved by tyrosine kinase inhibitors (TKIs such as imatinib mesylate (IM but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

  15. Solid renal tumor severity in von Hippel Lindau disease is related to germline deletion length and location.

    Science.gov (United States)

    Maranchie, Jodi K; Afonso, Anoushka; Albert, Paul S; Kalyandrug, Sivaram; Phillips, John L; Zhou, Shubo; Peterson, James; Ghadimi, Bijan M; Hurley, Katheen; Riss, Joseph; Vasselli, James R; Ried, Thomas; Zbar, Berton; Choyke, Peter; Walther, McClellan M; Klausner, Richard D; Linehan, W Marston

    2004-01-01

    von Hippel Lindau disease (VHL) is an autosomal dominant familial cancer syndrome linked to alteration of the VHL tumor suppressor gene. Affected patients are predisposed to develop pheochromocytomas and cystic and solid tumors of the kidney, CNS, pancreas, retina, and epididymis. However, organ involvement varies considerably among families and has been shown to correlate with the underlying germline alteration. Clinically, we observed a paradoxically lower prevalence of renal cell carcinoma (RCC) in patients with complete germline deletion of VHL. To determine if a relationship existed between the type of VHL deletion and disease, we retrospectively evaluated 123 patients from 55 families with large germline VHL deletions, including 42 intragenic partial deletions and 13 complete VHL deletions, by history and radiographic imaging. Each individual and family was scored for cystic or solid involvement of CNS, pancreas, and kidney, and for pheochromocytoma. Germline deletions were mapped using a combination of fluorescent in situ hybridization (FISH) and quantitative Southern and Southern blot analysis. An age-adjusted comparison demonstrated a higher prevalence of RCC in patients with partial germline VHL deletions relative to complete deletions (48.9 vs. 22.6%, p=0.007). This striking phenotypic dichotomy was not seen for cystic renal lesions or for CNS (p=0.22), pancreas (p=0.72), or pheochromocytoma (p=0.34). Deletion mapping revealed that development of RCC had an even greater correlation with retention of HSPC300 (C3orf10), located within the 30-kb region of chromosome 3p, immediately telomeric to VHL (52.3 vs. 18.9%, p <0.001), suggesting the presence of a neighboring gene or genes critical to the development and maintenance of RCC. Careful correlation of genotypic data with objective phenotypic measures will provide further insight into the mechanisms of tumor formation. Copyright 2003 Wiley-Liss, Inc.

  16. A Comprehensive tRNA Deletion Library Unravels the Genetic Architecture of the tRNA Pool

    Science.gov (United States)

    Bloom-Ackermann, Zohar; Navon, Sivan; Gingold, Hila; Towers, Ruth; Pilpel, Yitzhak; Dahan, Orna

    2014-01-01

    Deciphering the architecture of the tRNA pool is a prime challenge in translation research, as tRNAs govern the efficiency and accuracy of the process. Towards this challenge, we created a systematic tRNA deletion library in Saccharomyces cerevisiae, aimed at dissecting the specific contribution of each tRNA gene to the tRNA pool and to the cell's fitness. By harnessing this resource, we observed that the majority of tRNA deletions show no appreciable phenotype in rich medium, yet under more challenging conditions, additional phenotypes were observed. Robustness to tRNA gene deletion was often facilitated through extensive backup compensation within and between tRNA families. Interestingly, we found that within tRNA families, genes carrying identical anti-codons can contribute differently to the cellular fitness, suggesting the importance of the genomic surrounding to tRNA expression. Characterization of the transcriptome response to deletions of tRNA genes exposed two disparate patterns: in single-copy families, deletions elicited a stress response; in deletions of genes from multi-copy families, expression of the translation machinery increased. Our results uncover the complex architecture of the tRNA pool and pave the way towards complete understanding of their role in cell physiology. PMID:24453985

  17. Chromosomal deletion unmasking a recessive disease: 22q13 deletion syndrome and metachromatic leukodystrophy

    DEFF Research Database (Denmark)

    Bisgaard, A-M; Kirchhoff, M; Nielsen, J E

    2008-01-01

    A deletion on one chromosome and a mutant allele on the other may cause an autosomal recessive disease. We report on two patients with mental retardation, dysmorphic features and low catalytic activity of arylsulfatase A. One patient had a pathogenic mutation in the arylsulfatase A gene (ARSA......) and succumbed to metachromatic leukodystrophy (MLD). The other patient had a pseudoallele, which does not lead to MLD. The presenting clinical features and low arylsulfatase A activity were explained, in each patients, by a deletion of 22q13 and, thereby, of one allele of ARSA....

  18. B cell activation by outer membrane vesicles--a novel virulence mechanism.

    Directory of Open Access Journals (Sweden)

    Maria Laura A Perez Vidakovics

    2010-01-01

    Full Text Available Secretion of outer membrane vesicles (OMV is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR clustering and Ca(2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+ IgD(+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86, whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

  19. Writing and deleting single magnetic skyrmions.

    Science.gov (United States)

    Romming, Niklas; Hanneken, Christian; Menzel, Matthias; Bickel, Jessica E; Wolter, Boris; von Bergmann, Kirsten; Kubetzka, André; Wiesendanger, Roland

    2013-08-09

    Topologically nontrivial spin textures have recently been investigated for spintronic applications. Here, we report on an ultrathin magnetic film in which individual skyrmions can be written and deleted in a controlled fashion with local spin-polarized currents from a scanning tunneling microscope. An external magnetic field is used to tune the energy landscape, and the temperature is adjusted to prevent thermally activated switching between topologically distinct states. Switching rate and direction can then be controlled by the parameters used for current injection. The creation and annihilation of individual magnetic skyrmions demonstrates the potential for topological charge in future information-storage concepts.

  20. Quantitative Genome-Wide Analysis of Yeast Deletion Strain Sensitivities to Oxidative and Chemical Stress

    Directory of Open Access Journals (Sweden)

    Stanley Fields

    2006-03-01

    Full Text Available Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains (Winzeler et al., 1999. Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.

  1. The Role of Dicentric Chromosome Formation and Secondary Centromere Deletion in the Evolution of Myeloid Malignancy

    Science.gov (United States)

    MacKinnon, Ruth N.; Campbell, Lynda J.

    2011-01-01

    Dicentric chromosomes have been identified as instigators of the genome instability associated with cancer, but this instability is often resolved by one of a number of different secondary events. These include centromere inactivation, inversion, and intercentromeric deletion. Deletion or excision of one of the centromeres may be a significant occurrence in myeloid malignancy and other malignancies but has not previously been widely recognized, and our reports are the first describing centromere deletion in cancer cells. We review what is known about dicentric chromosomes and the mechanisms by which they can undergo stabilization in both constitutional and cancer genomes. The failure to identify centromere deletion in cancer cells until recently can be partly explained by the standard approaches to routine diagnostic cancer genome analysis, which do not identify centromeres in the context of chromosome organization. This hitherto hidden group of primary dicentric, secondary monocentric chromosomes, together with other unrecognized dicentric chromosomes, points to a greater role for dicentric chromosomes in cancer initiation and progression than is generally acknowledged. We present a model that predicts and explains a significant role for dicentric chromosomes in the formation of unbalanced translocations in malignancy. PMID:22567363

  2. The Role of Dicentric Chromosome Formation and Secondary Centromere Deletion in the Evolution of Myeloid Malignancy

    Directory of Open Access Journals (Sweden)

    Ruth N. MacKinnon

    2011-01-01

    Full Text Available Dicentric chromosomes have been identified as instigators of the genome instability associated with cancer, but this instability is often resolved by one of a number of different secondary events. These include centromere inactivation, inversion, and intercentromeric deletion. Deletion or excision of one of the centromeres may be a significant occurrence in myeloid malignancy and other malignancies but has not previously been widely recognized, and our reports are the first describing centromere deletion in cancer cells. We review what is known about dicentric chromosomes and the mechanisms by which they can undergo stabilization in both constitutional and cancer genomes. The failure to identify centromere deletion in cancer cells until recently can be partly explained by the standard approaches to routine diagnostic cancer genome analysis, which do not identify centromeres in the context of chromosome organization. This hitherto hidden group of primary dicentric, secondary monocentric chromosomes, together with other unrecognized dicentric chromosomes, points to a greater role for dicentric chromosomes in cancer initiation and progression than is generally acknowledged. We present a model that predicts and explains a significant role for dicentric chromosomes in the formation of unbalanced translocations in malignancy.

  3. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Science.gov (United States)

    Fernández, Esteban R; Olivera, Gabriela C; Quebrada Palacio, Luz P; González, Mariela N; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L; Ledesma Patiño, Oscar S; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  4. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Esteban R Fernández

    Full Text Available Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  5. Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury

    Science.gov (United States)

    Xie, Cuicui; Ginet, Vanessa; Sun, Yanyan; Koike, Masato; Zhou, Kai; Li, Tao; Li, Hongfu; Li, Qian; Wang, Xiaoyang; Uchiyama, Yasuo; Truttmann, Anita C.; Kroemer, Guido; Puyal, Julien; Blomgren, Klas; Zhu, Changlian

    2016-01-01

    ABSTRACT Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy. PMID:26727396

  6. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

    Science.gov (United States)

    2012-01-01

    Background Hepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients. Results Among the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone

  7. Deletion of ultraconserved elements yields viable mice

    Energy Technology Data Exchange (ETDEWEB)

    Ahituv, Nadav; Zhu, Yiwen; Visel, Axel; Holt, Amy; Afzal, Veena; Pennacchio, Len A.; Rubin, Edward M.

    2007-07-15

    Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lacking these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.

  8. Method for introducing unidirectional nested deletions

    Science.gov (United States)

    Dunn, J.J.; Quesada, M.A.; Randesi, M.

    1999-07-27

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector. The cloning vector has an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.

  9. Rare human diseases: 9p deletion syndrome

    Directory of Open Access Journals (Sweden)

    Galagan V.O.

    2014-09-01

    Full Text Available Objective of the study was to review the anamnesis, pheno - and genotype in patients with rare chromosome disorders such as 9p deletion syndrome. Genetic methods of investigation (clinical and genealogical, cytogenetic, FISH- method, paraclinical and instrumental methods of examination were used. Karyotyping was performed by the G-method of differential staining of chromosomes. Only three cases of pathology were diagnosed in the Medical Genetics Center over the last 10 years. By anamnesis data nobody in the probands’ families had bad habits, was exposed to occupational hazards, took part in the elimination of the Chernobyl accident or lived in contaminated areas. Clinical signs of diseases have not been identified in probands’ parents. All probands had trigonocephaly, bilateral epicanthal folds, ocular hypertelorism, downslanting palpebral fissures, long philtrum, flat face and nasal bridge, low set ears with malformed auricles. Two patients of three ones had exophthalmos, contracture of the second and third fingers, abnormal external genitalia. In all three cases there was monosomy of chromosome 9 of critical segment p 24. Normal karyotypes were seen in all parents, so there were three cases of new mutations of 9p deletion syndrome. Retardation of physical, psycho-spech, mental development in proband with or without congenital anomalies requires medical genetic counseling in a specialized institution. Cases of reproductive loss in anamnesis require cytogenetic investigation of fetal membranes and amniotic fluid.

  10. Brownfields/IGD: EF_ACRES

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_ACRES is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  11. Hazardous Waste/IGD: EF_RCRA

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_RCRA is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  12. Air Emissions/IGD: EF_AFS

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_AFS is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  13. Water Dischargers/IGD: EF_NPDES

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_NPDES is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  14. Superfund/IGD: EF_NPL

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_NPL is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  15. Toxic Releases/IGD: EF_TRI

    Data.gov (United States)

    U.S. Environmental Protection Agency — EF_TRI is a subset of facilities from FRS_INTEREST and FRS_FACILITY_SITE which are updated on a monthly basis as part of the Locational Reference Tables (LRT)...

  16. Are there ethnic differences in deletions in the dystrophin gene?

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, M.; Verma, I.C. [All India Inst. of Medical Sciences, New Delhi (India)

    1997-01-20

    We studied 160 cases of Duchenne muscular dystrophy (DMD) drawn from all parts of India, using multiplex PCR of 27 exons. Of these, 103 (64.4%) showed intragenic deletions. Most (69.7%) of the deletions involved exons 45-51. The phenotype of cases with deletion of single exons did not differ significantly from those with deletion of multiple exons. The distribution of deletions in studies from different countries was variable, but this was accounted for either by the small number of cases studied, or by fewer exons analyzed. It is concluded that there is likely to be no ethnic difference with respect to deletions in the DMD gene. 38 refs., 2 figs., 3 tabs.

  17. Panchromatic cooperative hyperspectral adaptive wide band deletion repair method

    Science.gov (United States)

    Jiang, Bitao; Shi, Chunyu

    2018-02-01

    In the hyperspectral data, the phenomenon of stripe deletion often occurs, which seriously affects the efficiency and accuracy of data analysis and application. Narrow band deletion can be directly repaired by interpolation, and this method is not ideal for wide band deletion repair. In this paper, an adaptive spectral wide band missing restoration method based on panchromatic information is proposed, and the effectiveness of the algorithm is verified by experiments.

  18. NPL deletion policy for RCRA-regulated TSD facilities finalized

    International Nuclear Information System (INIS)

    Anon.

    1995-01-01

    Under a new policy published by EPA on March 20, 1995, certain sites may be deleted from the National Priorities List (NPL) and deferred to RCRA corrective action. To be deleted from the NPL, a site must (1) be regulated under RCRA as a treatment, storage, or disposal (TSD) facility and (2) meet the four criteria specified by EPA. The new NPL deletion policy, which does not pertain to federal TSD facilities, became effective on April 19, 1995. 1 tab

  19. Clival encephalocele and 5q15 deletion: a case report.

    Science.gov (United States)

    Puvabanditsin, Surasak; Malik, Imran; Garrow, Eugene; Francois, Lissa; Mehta, Rajeev

    2015-03-01

    A preterm neonate presenting with respiratory distress after birth was found to have a clival encephalocele, which is a variant of a basal encephalocele, and hypoplasia of the cerebellum. Genetic studies revealed a small deletion of the long arm of chromosome 5: 5q15 deletion. We report a rare variant of a basal encephalocele with a cerebellar malformation and 5q15 deletion. © The Author(s) 2014.

  20. Male gametophytic sterility. 1 - Gametic sterilities and deletions in petunia

    Energy Technology Data Exchange (ETDEWEB)

    Cornu, A.; Maizonnier, D. (Station d' Amelioration des Plantes de l' I.N.R.A., Dijon (France))

    1982-01-01

    Terminal deletions induced by ionizing radiations in Petunia are not sexually transmitted. Cytogenetic study of plants with a heterozygous deletion and their progenies shows that this lack of transmission is accompanied by a gametic semi-sterility due to the fact that gametes carrying the deleted chromosome are not viable. The interest of such a male sterility with a gametophytic determinism for the study of sporophyte-gametophyte relationships is underlined.

  1. HOXA genes cluster: clinical implications of the smallest deletion

    OpenAIRE

    Pezzani, Lidia; Milani, Donatella; Manzoni, Francesca; Baccarin, Marco; Silipigni, Rosamaria; Guerneri, Silvana; Esposito, Susanna

    2015-01-01

    Background HOXA genes cluster plays a fundamental role in embryologic development. Deletion of the entire cluster is known to cause a clinically recognizable syndrome with mild developmental delay, characteristic facies, small feet with unusually short and big halluces, abnormal thumbs, and urogenital malformations. The clinical manifestations may vary with different ranges of deletions of HOXA cluster and flanking regions. Case presentation We report a girl with the smallest deletion reporte...

  2. SLUG (SNAI2) deletions in patients with Waardenburg disease.

    Science.gov (United States)

    Sánchez-Martín, Manuel; Rodríguez-García, Arancha; Pérez-Losada, Jesús; Sagrera, Ana; Read, Andrew P; Sánchez-García, Isidro

    2002-12-01

    Waardenburg syndrome (WS; deafness with pigmentary abnormalities) is a congenital disorder caused by defective function of the embryonic neural crest. Depending on additional symptoms, WS is classified into four types: WS1, WS2, WS3 and WS4. WS1 and WS3 are caused by mutations in PAX3, whereas WS2 is heterogenous, being caused by mutations in the microphthalmia (MITF) gene in some but not all affected families. The identification of Slugh, a zinc-finger transcription factor expressed in migratory neural crest cells, as the gene responsible for pigmentary disturbances in mice prompted us to analyse the role of its human homologue SLUG in neural crest defects. Here we show that two unrelated patients with WS2 have homozygous deletions in SLUG which result in absence of the SLUG product. We further show that Mitf is present in Slug-deficient cells and transactivates the SLUG promoter, and that Slugh and Kit genetically interact in vivo. Our findings further define the locus heterogeneity of WS2 and point to an essential role of SLUG in the development of neural crest-derived human cell lineages: its absence causes the auditory-pigmentary symptoms in at least some individuals with WS2.

  3. A Comparative Study of Quantum and Classical Deletion

    International Nuclear Information System (INIS)

    Shen Yao; Hao Liang; Long Guilu

    2010-01-01

    Here in this letter, we study the difference between quantum and classical deletion. We point out that the linear mapping deletion operation used in the impossibility proof for quantum systems applies also to classical system. The general classical deletion operation is a combined operation of measurement and transformation, i.e., first read the state and then transfer the state to the standard blank state. Though both quantum information and classical information can be deleted in an open system, quantum information cannot be recovered while classical information can be recovered. (general)

  4. Comprehensive analysis of pathogenic deletion variants in Fanconi anemia genes.

    Science.gov (United States)

    Flynn, Elizabeth K; Kamat, Aparna; Lach, Francis P; Donovan, Frank X; Kimble, Danielle C; Narisu, Narisu; Sanborn, Erica; Boulad, Farid; Davies, Stella M; Gillio, Alfred P; Harris, Richard E; MacMillan, Margaret L; Wagner, John E; Smogorzewska, Agata; Auerbach, Arleen D; Ostrander, Elaine A; Chandrasekharappa, Settara C

    2014-11-01

    Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution comparative genome hybridization arrays, single-nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants. © 2014 WILEY PERIODICALS, INC.

  5. 75 FR 1355 - Procurement List Additions and Deletions

    Science.gov (United States)

    2010-01-11

    .../Location: Janitorial Services, Jamestown Service Center, 8430 Country Club Street, Jamestown, ND. NPA..., the following products and services are deleted from the Procurement List: Products Business Cards NSN...

  6. High Prevalence of the BIM Deletion Polymorphism in Young Female Breast Cancer in an East Asian Country.

    Directory of Open Access Journals (Sweden)

    Ching-Hung Lin

    Full Text Available A rapid surge of female breast cancer has been observed in young women in several East Asian countries. The BIM deletion polymorphism, which confers cell resistance to apoptosis, was recently found exclusively in East Asian people with prevalence rate of 12%. We aimed to evaluate the possible role of this genetic alteration in carcinogenesis of breast cancer in East Asians.Female healthy volunteers (n = 307, patients in one consecutive stage I-III breast cancer cohort (n = 692 and one metastatic breast cancer cohort (n = 189 were evaluated. BIM wild-type and deletion alleles were separately genotyped in genomic DNAs.Both cancer cohorts consistently showed inverse associations between the BIM deletion polymorphism and patient age (≤35 y vs. 36-50 y vs. >50 y: 29% vs. 22% vs. 15%, P = 0.006 in the consecutive cohort, and 40% vs. 23% vs. 13%, P = 0.023 in the metastatic cohort. In healthy volunteers, the frequencies of the BIM deletion polymorphism were similar (13%-14% in all age groups. Further analyses indicated that the BIM deletion polymorphism was not associated with specific clinicopathologic features, but it was associated with poor overall survival (adjusted hazard ratio 1.71 in the consecutive cohort.BIM deletion polymorphism may be involved in the tumorigenesis of the early-onset breast cancer among East Asians.

  7. Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation

    International Nuclear Information System (INIS)

    Chen Xiaosui; Zhou Lijun; Wang Yuxiao; Qu Jia; Feng Jiangbing; Lu Xue; Chen Deqing; Liu Qingjie

    2006-01-01

    Objective: To construct a stable plasmid that spanning deleted human mitochondrial DNA (mtDNA) 4977 bp induced by ionizing radiation and another one for control DNA fragment, in order to use in the human mitochondrial genome study in the future. Methods: The peripheral blood, which had no mtDNA 4977 bp deletion found in previous study, was exposed to 10 Gy 60 Co γ-rays in vitro. The total cell DNA was extracted and PCR was carried out: a nest-PCR of three-round PCR was used for the mtDNA 4977 bp deletion and one- round regular PCR was used for the control ND1 gene. The PCR products were used for transfection by electroporation and the positive clones were obtained after screening. The plasmid DNA was isolated and sequenced after enzymatic digestion and purification. The sequence result was BLASTed with the human mitochondrial genome. Results: The sizes of PCR products for the flanked 4977 bp deletion and the ND1 gene were similar with those predicted according to GeneBank. The sequences for the positive clones were above 99 per cent homologous with the human mitochondrial genome after BLASTed. Conclusion: The plasmids for deleted human mtDNA 4977 bp and control DNA fragment have been constructed successfully, and they could be used in the quality and quantity studies on human mtDNA 4977 bp deletion. (authors)

  8. Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Sheila Xinxuan Soh

    Full Text Available A broad range of anti-cancer agents, including glucocorticoids (GCs and tyrosine kinase inhibitors (TKIs, kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL, we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.

  9. Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: deletion of the lon gene.

    Science.gov (United States)

    Leyman, Bregje; Boyen, Filip; Van Parys, Alexander; Verbrugghe, Elin; Haesebrouck, Freddy; Pasmans, Frank

    2012-12-01

    Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Mitochondrial DNA deletion and impairment of mitochondrial biogenesis are mediated by reactive oxygen species in ionizing radiation-induced premature senescence

    International Nuclear Information System (INIS)

    Eom, Hyeon Soo; Jung, U Hee; Jo, Sung Kee; Kim, Young Sang

    2011-01-01

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated β-galactosidase (SA-β-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and H 2 O 2 -treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and H 2 O 2 -treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-β-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

  11. Mitochondrial DNA deletion and impairment of mitochondrial biogenesis are mediated by reactive oxygen species in ionizing radiation-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyeon Soo; Jung, U Hee; Jo, Sung Kee [Radiation Biotechnology Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Young Sang [College of Natural Sciences, Chungnam National University, Daejeon (Korea, Republic of)

    2011-09-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated {beta}-galactosidase (SA-{beta}-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and H{sub 2}O{sub 2}-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and H{sub 2}O{sub 2}-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-{beta}-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

  12. Chromosomal deletion, promoter hypermethylation and downregulation of FYN in prostate cancer

    DEFF Research Database (Denmark)

    Sørensen, Karina Dalsgaard; Borre, Michael; Ørntoft, Torben Falck

    2008-01-01

    prostate hyperplasia (BPH), as well as in 6 prostate adenocarcinoma cell lines compared with that in BPH-1 cells. By immunohistochemistry, FYN protein was detected in nonmalignant prostate epithelium, but not in cancerous glands. Moreover, genomic bisulfite sequencing revealed frequent aberrant methylation......, consistent with gene silencing, was detected in 2 of 18 tumors (11%). No methylation was found in BPH-1 cells or nonmalignant prostate tissue samples (0 of 7). These results indicate that FYN is downregulated in prostate cancer by both chromosomal deletion and promoter hypermethylation, and therefore...

  13. Brand deletion: How the decision-making approach affects deletion success

    Directory of Open Access Journals (Sweden)

    Víctor Temprano-García

    2018-04-01

    Full Text Available Literature on brand deletion (BD, a critical and topical decision within a firm's marketing strategy, is extremely scarce. The present research is concerned with the decision-making process and examines the effect on BD success of three different approaches to decision-making – rational, intuitive and political – and of the interaction between the rational and political approaches. The moderating effect of the type of BD – i.e., total brand killing or disposal vs. brand name change – is also analyzed. The model is tested on a sample of 155 cases of BD. Results point to positive effects on BD success of both rationality and intuition, and a negative effect of politics. Findings also indicate that the negative impact of political behavior on BD success is minimized in the absence of evidence and objective information and when the BD is undertaken through a brand name change. JEL classification: L10, M31, Keywords: Brand deletion, Rational decision-making, Intuitive decision-making, Political decision-making, Brand deletion success

  14. Phosphatase and tensin homologue deleted on chromosome 10 ...

    African Journals Online (AJOL)

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH) for the PTEN ...

  15. Generalised deletion designs | Gachii | African Journal of Science ...

    African Journals Online (AJOL)

    In this paper asymmetrical single replicate factorial designs are constructed from symmetrical single replicate factorial designs using the deletion technique. The study is along the lines of Voss(1986), Chauhan(1989) and Gachii and Odhiambo(1997). We give results for the general order deletion designs of the form sn-m1(s ...

  16. 24 CFR 990.155 - Addition and deletion of units.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development Regulations Relating to Housing and Urban...; Computation of Eligible Unit Months § 990.155 Addition and deletion of units. (a) Changes in public housing...

  17. 4977-bp mitochondrial DNA deletion in infertile patients with varicocele.

    Science.gov (United States)

    Gashti, N G; Salehi, Z; Madani, A H; Dalivandan, S T

    2014-04-01

    Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977-bp deletion in infertile men with varicocele. To detect 4977-bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977-bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977-bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15-57.57, P deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977-bp deletion and varicocele-induced infertility, larger population-based studies are needed. It is concluded that there is an association between sperm mtDNA 4977-bp deletion and varicocele-induced infertility in the population studied. © 2013 Blackwell Verlag GmbH.

  18. 34 CFR 5.16 - Deletion of identifying details.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Deletion of identifying details. 5.16 Section 5.16 Education Office of the Secretary, Department of Education AVAILABILITY OF INFORMATION TO THE PUBLIC PURSUANT TO PUB. L. 90-23 (Eff. until 7-14-10) What Records Are Available § 5.16 Deletion of identifying...

  19. 42 CFR 401.118 - Deletion of identifying details.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 2 2010-10-01 2010-10-01 false Deletion of identifying details. 401.118 Section 401.118 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN... Deletion of identifying details. When CMS publishes or otherwise makes available an opinion or order...

  20. Coexistence of 9p Deletion Syndrome and Autism Spectrum Disorder

    Science.gov (United States)

    Günes, Serkan; Ekinci, Özalp; Ekinci, Nuran; Toros, Fevziye

    2017-01-01

    Deletion or duplication of the short arm of chromosome 9 may lead to a variety of clinical conditions including craniofacial and limb abnormalities, skeletal malformations, mental retardation, and autism spectrum disorder. Here, we present a case report of 5-year-old boy with 9p deletion syndrome and autism spectrum disorder.

  1. Linguistic and Psychomotor Development in Children with Chromosome 14 Deletions

    Science.gov (United States)

    Zampini, Laura; D'Odorico, Laura; Zanchi, Paola; Zollino, Marcella; Neri, Giovanni

    2012-01-01

    The present study focussed on a specific type of rare genetic condition: chromosome 14 deletions. Children with this genetic condition often show developmental delays and brain and neurological problems, although the type and severity of symptoms varies depending on the size and location of the deleted genetic material. The specific aim of the…

  2. 76 FR 78248 - Procurement List; Addition and Deletions

    Science.gov (United States)

    2011-12-16

    .... Service Type/Location: Laundry Service, Stratton Medical Center, 113 Holland Ave, Albany, NY. [[Page 78249...: Addition to and Deletions from the Procurement List. SUMMARY: This action adds a service to the Procurement... disabilities, and deletes products and services from the Procurement List previously furnished by such agencies...

  3. 75 FR 49481 - Procurement List; Additions and Deletion

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    2010-08-13

    ... added to the Procurement List: Services Service Type/Locations: Laundry Service, Atlanta VA Medical...: Additions to and deletion from the Procurement List. SUMMARY: This action adds services to the Procurement... disabilities and deletes a service from the Procurement List previously furnished by such agency. DATES...

  4. 78 FR 21916 - Procurement List; Addition And Deletions

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    2013-04-12

    ..., the following service is added to the Procurement List: Service Service Type/Location: Laundry Service...: Addition to and Deletions from the Procurement List. SUMMARY: This action adds a service to the Procurement... disabilities, and deletes products and services from the Procurement List previously furnished by such agencies...

  5. 78 FR 53733 - Procurement List Additions and Deletions

    Science.gov (United States)

    2013-08-30

    .../Location: Industrial Laundry Service, Bureau of Engraving and Printing, 9000 Blue Mound Road, Fort Worth...: Additions to and Deletions from the Procurement List. SUMMARY: This action adds products and services to the... severe disabilities, and deletes services from the Procurement List previously provided by such agencies...

  6. Recurrence and Variability of Germline EPCAM Deletions in Lynch Syndrome

    NARCIS (Netherlands)

    Kuiper, Roland P.; Vissers, Lisenka E. L. M.; Venkatachalam, Ramprasath; Bodmer, Danielle; Hoenselaar, Eveline; Goossens, Monique; Haufe, Aline; Kamping, Eveline; Niessen, Renee C.; Hogervorst, Frans B. L.; Gille, Johan J. P.; Redeker, Bert; Tops, Carli M. J.; van Gijn, Marielle E.; van den Ouweland, Ans M. W.; Rahner, Nils; Steinke, Verena; Kahl, Philip; Holinski-Feder, Elke; Morak, Monika; Kloor, Matthias; Stemmler, Susanne; Betz, Beate; Hutter, Pierre; Bunyan, David J.; Syngal, Sapna; Culver, Julie O.; Graham, Tracy; Chan, Tsun L.; Nagtegaal, Iris D.; van Krieken, J. Han J. M.; Schackert, Hans K.; Hoogerbrugge, Nicoline; van Kessel, Ad Geurts; Ligtenberg, Marjolijn J. L.

    Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like

  7. Clonal deletion: A mechanism of tolerance in mixed bone marrow chimeras

    International Nuclear Information System (INIS)

    Yu, J.C.; Webster, M.; Fox, I.J.

    1990-01-01

    The mechanism of antigen-specific immunologic unresponsiveness which results from lethal irradiation and mixed (syngeneic-allogeneic) bone marrow cell (BMC) reconstitution is unknown. To determine whether clonal deletion is the mechanism of tolerance in this model, monoclonal antibody (Mab) RR-4-4, specific for a T-cell receptor (V beta 6) reactive against the minor alloantigen MLsa, was employed. Six-week-old B10 mice (H-2b, Mlsb, Thyl.2) were tolerized to AKR antigens (H-2k, Mlsa, Thyl.1) by whole body irradiation (950 R) and iv infusion of T-cell-depleted (TCD) B10 BMC + non-TCD AKR BMC. Chimerism and antigen-specific tolerance were documented by flow microfluorometry (FMF), skin grafting, mixed lymphocyte reaction, and cell-mediated lympholysis. When tolerant B10 mice (n = 15) had accepted AKR skin grafts for greater than 100 days, these animals were studied for the presence of host V beta 6+ T cells using Mab RR-4-4. FMF revealed that 0-5% of host (B10) lymph node and spleen cells from chimeras were V beta 6+ while 15-20% of lymph node and spleen cells from control B10 mice expressed V beta 6. These data demonstrate that clonal deletion occurs in the lethal irradiation-mixed reconstitution model as evidenced by the near total elimination of Mlsa-reactive V beta 6+ T cells and suggest that it maybe a mechanism responsible for tolerance in adult mice

  8. Comprehensive detection of diverse exon 19 deletion mutations of EGFR in lung Cancer by a single probe set.

    Science.gov (United States)

    Bae, Jin Ho; Jo, Seong-Min; Kim, Hak-Sung

    2015-12-15

    Detection of exon 19 deletion mutation of EGFR, one of the most frequently occurring mutations in lung cancer, provides the crucial information for diagnosis and treatment guideline in non-small-cell lung cancer (NSCLC). Here, we demonstrate a simple and efficient method to detect various exon 19 deletion mutations of EGFR using a single probe set comprising of an oligo-quencher (oligo-Q) and a molecular beacon (MB). While the MB hybridizes to both the wild and mutant target DNA, the oligo-Q only binds to the wild target DNA, leading to a fluorescent signal in case of deletion mutation. This enables the comprehensive detection of the diverse exon 19 deletion mutations using a single probe set. We demonstrated the utility and efficiency of the approach by detecting the frequent exon 19 deletion mutations of EGFR through a real-time PCR and in situ fluorescence imaging. Our approach enabled the detection of genomic DNA as low as 0.02 ng, showing a detection limit of 2% in a heterogeneous DNA mixture, and could be used for detecting mutations in a single cell level. The present MB and oligo-Q dual probe system can be used for diagnosis and treatment guideline in NSCLC. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Kidney epithelium specific deletion of kelch-like ECH-associated protein 1 (Keap1) causes hydronephrosis in mice.

    Science.gov (United States)

    Noel, Sanjeev; Arend, Lois J; Bandapalle, Samatha; Reddy, Sekhar P; Rabb, Hamid

    2016-08-02

    Transcription factor Nrf2 protects from experimental acute kidney injury (AKI) and is promising to limit progression in human chronic kidney disease (CKD) by upregulating multiple antioxidant genes. We recently demonstrated that deletion of Keap1, the endogenous inhibitor of Nrf2, in T lymphocytes significantly protects from AKI. In this study, we investigated the effect of Keap1 deletion on Nrf2 mediated antioxidant response in the renal tubular epithelial cells. We deleted Keap1 exon 2 and 3 in the renal tubular epithelial cells by crossing Ksp-Cre mice with Keap1 floxed (Keap1 (f/f)) mice. Deletion of Keap1 gene in the kidney epithelial cells of Ksp-Keap1 (-/-) mice and its effect on Nrf2 target gene expression was performed using PCR and real-time PCR respectively. Histological evaluation was performed on H&E stained sections. Complete blood count, serum and urine analysis were performed to assess systemic effects of defective kidney development. Student's T test was used to determine statistical difference between the groups. Ksp-Cre resulted in the deletion of Keap1 exon 2 and 3 and subsequent upregulation of Nrf2 target genes, Nqo1, Gclm and Gclc in the kidney epithelial cells of Ksp-Keap1 (-/-) mice at baseline. Renal epithelial cell specific deletion of Keap1 in Ksp-Keap1 (-/-) mice caused marked renal pelvic expansion and significant compression of medullary parenchyma consistent with hydronephrosis in both (3 month-old) males and females. Kidneys from 6 month-old Ksp-Keap1 (-/-) mice showed progressive hydronephrosis. Hematological, biochemical and urinary analysis showed significantly higher red blood cell count (p = 0.04), hemoglobin (p = 0.01), hematocrit (p = 0.02), mean cell volume (p = 0.02) and mean cell hemoglobin concentration (p = 0.003) in Ksp-Keap1 (-/-) mice in comparison to Keap1 (f/f) mice. These unexpected findings demonstrate that Keap1 deletion in renal tubular epithelial cells results in an abnormal kidney

  10. Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

    Science.gov (United States)

    Rocha, Mariana C.; Rosa, Hannah S.; Grady, John P.; Blakely, Emma L.; He, Langping; Romain, Nadine; Haller, Ronald G.; Newman, Jane; McFarland, Robert; Ng, Yi Shiau; Gorman, Grainne S.; Schaefer, Andrew M.; Tuppen, Helen A.; Taylor, Robert W.

    2018-01-01

    Objective Single, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle. Methods We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large‐scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. Results We have defined 3 “classes” of single, large‐scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. Interpretation Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex‐specific protein‐encoding genes. Furthermore, removal of mt‐tRNA genes impacts specific complexes only at high deletion levels, when complex‐specific protein‐encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115–130 PMID:29283441

  11. A Catalog of Genes Homozygously Deleted in Human Lung Cancer and the Candidacy of PTPRD as a Tumor Suppressor Gene

    Science.gov (United States)

    Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D.; Yokota, Jun

    2010-01-01

    A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. PMID:20073072

  12. Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo.

    Science.gov (United States)

    Pudupakam, R S; Huang, Y W; Opriessnig, T; Halbur, P G; Pierson, F W; Meng, X J

    2009-01-01

    Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.

  13. Fshb-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage.

    Science.gov (United States)

    Wang, Huizhen; Hastings, Richard; Miller, William L; Kumar, T Rajendra

    2016-01-05

    Genetic analysis of development and function of the gonadotrope cell lineage within mouse anterior pituitary has been greatly facilitated by at least three currently available Cre strains in which Cre was either knocked into the Gnrhr locus or expressed as a transgene from Cga and Lhb promoters. However, in each case there are some limitations including CRE expression in thyrotropes within pituitary or ectopic expression outside of pituitary, for example in some populations of neurons or gonads. Hence, these Cre strains often pose problems with regard to undesirable deletion of alleles in non-gonadotrope cells, fertility and germline transmission of mutant alleles. Here, we describe generation and characterization of a new Fshb-iCre deleter strain using 4.7 kb of ovine Fshb promoter regulatory sequences driving iCre expression exclusively in the gonadotrope lineage within anterior pituitary. Fshb-iCre mice develop normally, display no ectopic CRE expression in gonads and are fertile. When crossed onto a loxP recombination-mediated red to green color switch reporter mouse genetic background, in vivo CRE recombinase activity is detectable in gonadotropes at more than 95% efficiency and the GFP-tagged gonadotropes readily purified by fluorescence activated cell sorting. We demonstrate the applicability of this Fshb-iCre deleter strain in a mouse model in which Dicer is efficiently and selectively deleted in gonadotropes. We further show that loss of DICER-dependent miRNAs in gonadotropes leads to profound suppression of gonadotropins resulting in male and female infertility. Thus, Fshb-iCre mice serve as a new genetic tool to efficiently manipulate gonadotrope-specific gene expression in vivo. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Detection of genomic deletions in rice using oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Bordeos Alicia

    2009-03-01

    Full Text Available Abstract Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL. However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/. Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a

  15. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (T FH ) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of T FH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on T FH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate T FH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing T FH cell maturation. In cocultures they differentiated B cells into CD138 + plasma and IgD - CD27 + memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented T FH cell development. Added to T FH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3 + CXCR5 + PD-1 + follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on T FH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control T FH cell maturation, expand follicular regulatory T cells, and inhibit the T FH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the T FH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  16. Tau deletion promotes brain insulin resistance.

    Science.gov (United States)

    Marciniak, Elodie; Leboucher, Antoine; Caron, Emilie; Ahmed, Tariq; Tailleux, Anne; Dumont, Julie; Issad, Tarik; Gerhardt, Ellen; Pagesy, Patrick; Vileno, Margaux; Bournonville, Clément; Hamdane, Malika; Bantubungi, Kadiombo; Lancel, Steve; Demeyer, Dominique; Eddarkaoui, Sabiha; Vallez, Emmanuelle; Vieau, Didier; Humez, Sandrine; Faivre, Emilie; Grenier-Boley, Benjamin; Outeiro, Tiago F; Staels, Bart; Amouyel, Philippe; Balschun, Detlef; Buee, Luc; Blum, David

    2017-08-07

    The molecular pathways underlying tau pathology-induced synaptic/cognitive deficits and neurodegeneration are poorly understood. One prevalent hypothesis is that hyperphosphorylation, misfolding, and fibrillization of tau impair synaptic plasticity and cause degeneration. However, tau pathology may also result in the loss of specific physiological tau functions, which are largely unknown but could contribute to neuronal dysfunction. In the present study, we uncovered a novel function of tau in its ability to regulate brain insulin signaling. We found that tau deletion leads to an impaired hippocampal response to insulin, caused by altered IRS-1 and PTEN (phosphatase and tensin homologue on chromosome 10) activities. Our data also demonstrate that tau knockout mice exhibit an impaired hypothalamic anorexigenic effect of insulin that is associated with energy metabolism alterations. Consistently, we found that tau haplotypes are associated with glycemic traits in humans. The present data have far-reaching clinical implications and raise the hypothesis that pathophysiological tau loss-of-function favors brain insulin resistance, which is instrumental for cognitive and metabolic impairments in Alzheimer's disease patients. © 2017 Marciniak et al.

  17. Usefulness of MLPA in the detection of SHOX deletions.

    Science.gov (United States)

    Funari, Mariana F A; Jorge, Alexander A L; Souza, Silvia C A L; Billerbeck, Ana E C; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

    2010-01-01

    SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3'M'34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  18. Conversion of Deletions during Recombination in Pneumococcal Transformation

    Science.gov (United States)

    Lefevre, J. C.; Mostachfi, P.; Gasc, A. M.; Guillot, E.; Pasta, F.; Sicard, M.

    1989-01-01

    Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems. PMID:2599365

  19. A strong deletion bias in nonallelic gene conversion.

    Directory of Open Access Journals (Sweden)

    Raquel Assis

    Full Text Available Gene conversion is the unidirectional transfer of genetic information between orthologous (allelic or paralogous (nonallelic genomic segments. Though a number of studies have examined nucleotide replacements, little is known about length difference mutations produced by gene conversion. Here, we investigate insertions and deletions produced by nonallelic gene conversion in 338 Drosophila and 10,149 primate paralogs. Using a direct phylogenetic approach, we identify 179 insertions and 614 deletions in Drosophila paralogs, and 132 insertions and 455 deletions in primate paralogs. Thus, nonallelic gene conversion is strongly deletion-biased in both lineages, with almost 3.5 times as many conversion-induced deletions as insertions. In primates, the deletion bias is considerably stronger for long indels and, in both lineages, the per-site rate of gene conversion is orders of magnitudes higher than that of ordinary mutation. Due to this high rate, deletion-biased nonallelic gene conversion plays a key role in genome size evolution, leading to the cooperative shrinkage and eventual disappearance of selectively neutral paralogs.

  20. Deficiency in memory B cell compartment in a patient with infertility and recurrent pregnancy losses.

    Science.gov (United States)

    Sung, N; Byeon, H J; Garcia, M D Salazar; Skariah, A; Wu, L; Dambaeva, S; Beaman, K; Gilman-Sachs, A; Kwak-Kim, J

    2016-11-01

    Alterations in normal balance of B cell subsets have been reported in various rheumatic diseases. In this study, we report a woman with a history of recurrent pregnancy losses (RPL) and infertility who had low levels of memory B cells. A 35-year-old woman with a history of RPL and infertility was demonstrated to have increased peripheral blood CD19+ B cells with persistently low levels of memory B cell subsets. Prior to the frozen donor egg transfer cycle, prednisone and intravenous immunoglobulin G (IVIg) treatment was initiated and patient achieved dichorionic diamniotic twin pregnancies. During pregnancy, proportion (%) of switched memory B cells CD27+IgD- increased, while percent of total CD19+ B cells and CD27-IgD+ naive B cells were gradually decreased with a high dose IVIg treatment. She developed cervical incompetence at 20 weeks of gestation, received a Cesarean section at 32 weeks of gestation due to preterm labor, and delivered twin babies. B cell subset abnormalities may be associated with infertility, RPL and preterm labor, and further investigation is needed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Microarray data analyses of yeast RNA Pol I subunit RPA12 deletion strain

    Directory of Open Access Journals (Sweden)

    Kamlesh Kumar Yadav

    2016-06-01

    Full Text Available The ribosomal RNA (rRNA biosynthesis is the most energy consuming process in all living cells and the majority of total transcription activity is dedicated for synthesizing rRNA. The cells may adjust the synthesis of rRNA with the availability of resources. rRNA is mainly synthesized by RNA polymerase I that is composed of 14 subunits. Deletion of RPA12, 14, 39 and 49 are viable. RPA12 is a very small protein (13.6 kDa, and the amount of protein in the cells is very high (12,000 molecules per cell, but the role of this protein is unknown in other cellular metabolic processes (Kulak et al., 2014 [1]. RPA12 consists of two zinc-binding domains and it is required for the termination of rRNA synthesis (Mullem et al., 2002 [2]. Deletions of RPA12 in Saccharomyces cerevisiae and Schizosaccharomyces pombe cause a conditional growth defect (Nogi et al., 1993 [3]. In S. pombe, C-terminal deletion behaves like wild-type (Imazawa et al., 2001 [4]. This prompted us to investigate in detail the physiological role of RPA12 in S. cerevisiae, we performed the microarray of rpa12∆ strain and deposited into Gene Expression Omnibus under GSE68731. The analysis of microarray data revealed that the expression of major cellular metabolism genes is high. The amino acid biosynthesis, nonpolar lipid biosynthesis and glucose metabolic genes are highly expressed. The analyses also revealed that the rpa12∆ cells have an uncontrolled synthesis of cell metabolites, so RPA12 could be a master regulator for whole cellular metabolism.

  2. Performance of quantum cloning and deleting machines over coherence

    Science.gov (United States)

    Karmakar, Sumana; Sen, Ajoy; Sarkar, Debasis

    2017-10-01

    Coherence, being at the heart of interference phenomena, is found to be an useful resource in quantum information theory. Here we want to understand quantum coherence under the combination of two fundamentally dual processes, viz., cloning and deleting. We found the role of quantum cloning and deletion machines with the consumption and generation of quantum coherence. We establish cloning as a cohering process and deletion as a decohering process. Fidelity of the process will be shown to have connection with coherence generation and consumption of the processes.

  3. Brand deletion: How the decision-making approach affects deletion success

    OpenAIRE

    Víctor Temprano-García; Ana Isabel Rodríguez-Escudero; Javier Rodríguez-Pinto

    2018-01-01

    Literature on brand deletion (BD), a critical and topical decision within a firm's marketing strategy, is extremely scarce. The present research is concerned with the decision-making process and examines the effect on BD success of three different approaches to decision-making – rational, intuitive and political – and of the interaction between the rational and political approaches. The moderating effect of the type of BD – i.e., total brand killing or disposal vs. brand name change – is also...

  4. PTEN C-Terminal Deletion Causes Genomic Instability and Tumor Development

    Directory of Open Access Journals (Sweden)

    Zhuo Sun

    2014-03-01

    Full Text Available Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here, we describe a knockin mouse model of a nonsense mutation that results in the deletion of the entire Pten C-terminal region, referred to as PtenΔC. Mice heterozygous for PtenΔC develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C-terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing the initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C terminus in the maintenance of genomic stability and suppression of tumorigenesis.

  5. Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus.

    Science.gov (United States)

    Kong, Byung-Whi; Foster, Linda K; Foster, Douglas N

    2008-03-01

    The avian metapneumovirus (AMPV) genome encodes the fusion (F), small hydrophobic (SH), and attachment glycoprotein (G) as envelope glycoproteins. The F and G proteins mainly function to allow viral entry into host cells during the early steps of the virus life cycle. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes. Sequence analysis was used to determine if any differences between avian or mammalian cell propagated subtype C AMPV could be detected for the 1.8kb G gene. As a result, the complete 1.8kb G gene was found to be present when AMPV was propagated in our immortal turkey turbinate (TT-1) cell line regardless of passage number. Surprisingly, AMPV propagated for 15 or more passages in mammalian Vero cells revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. Although the Vero cell propagated AMPV genome contained a small 122 nucleotide fragment of the G gene, no other mRNA variants were detected from either mammalian or avian propagated AMPV. The G gene truncation might be caused by cellular molecular mechanisms that are species-specific. The lack of viral gene deletions suggests that avian cell propagated AMPV will provide a better alternative host for live recombinant vaccine development based on a reverse genetics system.

  6. Deleted in Malignant Brain Tumors 1 is Present in the Vascular Extracellular Matrix and Promotes Angiogenesis

    DEFF Research Database (Denmark)

    Müller-Enbergs, Helmut; Hu, Jiong; Popp, Rüdiger

    2012-01-01

    OBJECTIVE: Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. Here we studied the role of DMBT1 in endothelial cells. METHODS AND RESULTS: DMBT1 was secreted ...... and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4....

  7. Deletion of Dictyostelium discoideum Sir2A impairs cell proliferation ...

    Indian Academy of Sciences (India)

    Rakhee Lohia

    2018-04-13

    Apr 13, 2018 ... Multicellular structures developed were collected, fixed and stained with X-gal as ... classification for Sirtuins (Fyre 2000), the above were clas- ... RNA, protein and lipid substrates (Klug 1999; Hall 2005;. Gamsjaeger et al.

  8. Deleted in Malignant Brain Tumors 1 is up-regulated in bacterial endocarditis and binds to components of vegetations

    DEFF Research Database (Denmark)

    Müller, Hanna; Renner, Marcus; Helmke, Burkhard M

    2009-01-01

    OBJECTIVE: Bacterial endocarditis is a frequent infectious cardiac disease, especially in patients with congenital or acquired heart defects. It is characterized by bacterial colonization of the heart valves and the appearance of vegetations consisting of fibrin, blood cells, and bacteria....... The glycoprotein Deleted in Malignant Brain Tumors 1 is a scavenger receptor cysteine-rich protein with functions in innate immunity and epithelial differentiation. Because of the aggregating capacity of Deleted in Malignant Brain Tumors 1, we hypothesized that an up-regulation in bacterial endocarditis may...... be linked to the development of vegetations. METHODS: Heart tissue of 19 patients with bacterial endocarditis and 10 controls without bacterial endocarditis was analyzed by immunohistochemistry. The effect of human recombinant Deleted in Malignant Brain Tumors 1 on erythrocyte aggregation was measured using...

  9. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Ayesha; Ellenson, Lora Hedrick, E-mail: lora.ellenson@med.cornell.edu

    2011-07-01

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten{sup +/-} mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten{sup +/-} mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ER{alpha} as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  10. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    International Nuclear Information System (INIS)

    Joshi, Ayesha; Ellenson, Lora Hedrick

    2011-01-01

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten +/- mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten +/- mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  11. Genotype-Phenotype Analysis, Neuropsychological Assessment, and Growth Hormone Response in a Patient with 18p Deletion Syndrome.

    Science.gov (United States)

    Sun, Huihui; Wan, Naijun; Wang, Xinli; Chang, Liang; Cheng, Dazhi

    2018-01-01

    18p deletion syndrome is a rare chromosomal disease caused by deletion of the short arm of chromosome 18. By using cytogenetic and SNP array analysis, we identified a girl with 18p deletion syndrome exhibiting craniofacial anomalies, intellectual disability, and short stature. G-banding analysis of metaphase cells revealed an abnormal karyotype 46,XX,del(18)(p10). Further, SNP array detected a 15.3-Mb deletion at 18p11.21p11.32 (chr18:12842-15375878) including 61 OMIM genes. Genotype-phenotype correlation analysis showed that clinical manifestations of the patient were correlated with LAMA1, TWSG1, and GNAL deletions. Her neuropsychological assessment test demonstrated delay in most cognitive functions including impaired mathematics, linguistic skills, visual motor perception, respond speed, and executive function. Meanwhile, her integrated visual and auditory continuous performance test (IVA-CPT) indicated a severe comprehensive attention deficit. At age 7 and 1/12 years, her height was 110.8 cm (-2.5 SD height for age). Growth hormone (GH) treatment was initiated. After 27 months treatment, her height was increased to 129.6 cm (-1.0 SD height for age) at 9 and 4/12 years, indicating an effective response to GH treatment. © 2018 S. Karger AG, Basel.

  12. 22q11.2 deletion syndrome

    Science.gov (United States)

    McDonald-McGinn, Donna M.; Sullivan, Kathleen E.; Marino, Bruno; Philip, Nicole; Swillen, Ann; Vorstman, Jacob A. S.; Zackai, Elaine H.; Emanuel, Beverly S.; Vermeesch, Joris R.; Morrow, Bernice E.; Scambler, Peter J.; Bassett, Anne S.

    2016-01-01

    22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion disorder, estimated to result mainly from de novo non-homologous meiotic recombination events occurring in approximately 1 in every 1,000 fetuses. The first description in the English language of the constellation of findings now known to be due to this chromosomal difference was made in the 1960s in children with DiGeorge syndrome, who presented with the clinical triad of immunodeficiency, hypoparathyroidism and congenital heart disease. The syndrome is now known to have a heterogeneous presentation that includes multiple additional congenital anomalies and later-onset conditions, such as palatal, gastrointestinal and renal abnormalities, autoimmune disease, variable cognitive delays, behavioural phenotypes and psychiatric illness — all far extending the original description of DiGeorge syndrome. Management requires a multidisciplinary approach involving paediatrics, general medicine, surgery, psychiatry, psychology, interventional therapies (physical, occupational, speech, language and behavioural) and genetic counselling. Although common, lack of recognition of the condition and/or lack of familiarity with genetic testing methods, together with the wide variability of clinical presentation, delays diagnosis. Early diagnosis, preferably prenatally or neonatally, could improve outcomes, thus stressing the importance of universal screening. Equally important, 22q11.2DS has become a model for understanding rare and frequent congenital anomalies, medical conditions, psychiatric and developmental disorders, and may provide a platform to better understand these disorders while affording opportunities for translational strategies across the lifespan for both patients with 22q11.2DS and those with these associated features in the general population. PMID:27189754

  13. The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation

    Directory of Open Access Journals (Sweden)

    Hütter Gero

    2011-10-01

    Full Text Available Abstract Background The natural function of the C-C chemokine receptor type 5 (CCR5 is poorly understood. A 32 base pair deletion in the CCR5 gene (CCR5-delta32 located on chromosome 3 results in a non-functional protein. It is supposed that this deletion causes an alteration in T-cell response to inflammation. For example, the presence of the CCR5-delta32 allele in recipients of allografts constitutes as an independent and protective factor associated with a decreased risk of graft-versus-host disease (GVHD and graft rejection. However, the mechanism of this beneficial effect of the deletion regarding GVHD is unknown. In this survey we searched for a CCR5-delta32 associated regulation of critical genes involved in the immune response and the development of GVHD. Methods We examined CD34+ hematopoietic progenitor cells derived from bone marrow samples from 19 healthy volunteers for the CCR5-delta32 deletion with a genomic PCR using primers flanking the site of the deletion. Results 12 individuals were found to be homozygous for CCR5 WT and 7 carried the CCR5-delta32 deletion heterozygously. Global gene expression analysis led to the identification of 11 differentially regulated genes. Six of them are connected with mechanisms of immune response and control: LRG1, CXCR2, CCRL2, CD6, CD7, WD repeat domain, and CD30L. Conclusions Our data indicate that the CCR5-delta32 mutation may be associated with differential gene expression. Some of these genes are critical for immune response, in the case of CD30L probably protective in terms of GVHD.

  14. Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

    Science.gov (United States)

    Wang, Yi; Chen, Zhenmin; Zhao, Ruili; Jin, Tingting; Zhang, Xiaoming; Chen, Xiangdong

    2014-08-31

    Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

  15. Multigene deletions in lung adenocarcinomas from irradiated and control mice

    International Nuclear Information System (INIS)

    Zhang, Y.; Woloschak, G.E.

    1996-01-01

    K-ras codon 12 point mutations mRb and p53 gene deletions were examined in tissues from 120 normal lungs and lung adenocarcinomas that were Formalin-treated and paraffin-embedded 25 years ago. The results showed that 12 of 60 (20%) lung adenocarcinomas had mRb deletions. All lung adenocarcinomas that were initially found bearing deleted mRb had p53 deletions (15 of 15; 100%). A significantly higher mutation frequency for K-ras codon 12 point mutations was also found in the lung adenocarcinomas from mice exposed to 24 once-weekly neutron irradiation (10 of 10; 100%) compared with those exposed to 24 or 60 once-weekly γ-ray doses (5 of 10; 50%). The data suggested that p53 and K-ras gene alterations were two contributory factors responsible for the increased incidence of lung adenocarcinoma in B6CF 1 male mice exposed to protracted neutron radiation

  16. Additions and deletions to the known cerambycidae (Coleoptera) of Bolivia

    Science.gov (United States)

    An additional 137 species and two tribes are added to the known cerambycid fauna of Bolivia while 12 species are deleted. Comments and statistics regarding the growth of knowledge on the Bolivian Cerambycid fauna and species endemicity are included....

  17. 78 FR 75911 - Procurement List; Proposed Additions and Deletions

    Science.gov (United States)

    2013-12-13

    ... persons who are blind or have other severe disabilities and to delete products and a service previously...: General Services Administration, New York, NY NSN: 8955-01-E61-3689--Coffee, Roasted, Ground, 39 oz. bag...

  18. 76 FR 37069 - Procurement List; Proposed Additions and Deletion

    Science.gov (United States)

    2011-06-24

    ... Certification The following products and service are proposed for addition to Procurement List for production by... following product is proposed for deletion from the Procurement List: Product Detergent, Laundry NSN: 7930...

  19. Fast detection of deletion breakpoints using quantitative PCR

    Directory of Open Access Journals (Sweden)

    Gulshara Abildinova

    2016-01-01

    Full Text Available Abstract The routine detection of large and medium copy number variants (CNVs is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories.

  20. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  1. APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

    Science.gov (United States)

    Chen, Ting-Wen; Lee, Chi-Ching; Liu, Hsuan; Wu, Chi-Sheng; Pickering, Curtis R; Huang, Po-Jung; Wang, Jing; Chang, Ian Yi-Feng; Yeh, Yuan-Ming; Chen, Chih-De; Li, Hsin-Pai; Luo, Ji-Dung; Tan, Bertrand Chin-Ming; Chan, Timothy En Haw; Hsueh, Chuen; Chu, Lichieh Julie; Chen, Yi-Ting; Zhang, Bing; Yang, Chia-Yu; Wu, Chih-Ching; Hsu, Chia-Wei; See, Lai-Chu; Tang, Petrus; Yu, Jau-Song; Liao, Wei-Chao; Chiang, Wei-Fan; Rodriguez, Henry; Myers, Jeffrey N; Chang, Kai-Ping; Chang, Yu-Sun

    2017-09-06

    Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/- :89A3B +/- :27A3B +/+ ), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

  2. Two novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia (FH Siracusa and FH Reggio Emilia).

    Science.gov (United States)

    Garuti, R; Lelli, N; Barozzini, M; Tiozzo, R; Ghisellini, M; Simone, M L; Li Volti, S; Garozzo, R; Mollica, F; Vergoni, W; Bertolini, S; Calandra, S

    1996-03-01

    In the present study we report two novel partial deletions of the LDL-R gene. The first (FH Siracusa), found in an FH-heterozygote, consists of a 20 kb deletion spanning from the 5' flanking region to the intron 2 of the LDL-receptor gene. The elimination of the promoter and the first two exons prevents the transcription of the deleted allele, as shown by Northern blot analysis of LDL-R mRNA isolated from the proband's fibroblasts. The second deletion (FH Reggio Emilia), which eliminates 11 nucleotides of exon 10, was also found in an FH heterozygote. The characterization of this deletion was made possible by a combination of techniques such as single strand conformation polymorphism (SSCP) analysis, direct sequence of exon 10 and cloning of the normal and deleted exon 10 from the proband's DNA. The 11 nt deletion occurs in a region of exon 10 which contains three triplets (CTG) and two four-nucleotides (CTGG) direct repeats. This structural feature might render this region more susceptible to a slipped mispairing during DNA duplication. Since this deletion causes a shift of the BamHI site at the 5' end of exon 10, a method has been devised for its rapid screening which is based on the PCR amplification of exon 10 followed by BamHI digestion. FH Reggio Emilia deletion produces a shift in the reading frame downstream from Lys458, leading to a sequence of 51 novel amino acids before the occurrence of a premature stop codon (truncated receptor). However, since RT-PCR failed to demonstrate the presence of the mutant LDL-R mRNA in proband fibroblasts, it is likely that the amount of truncated receptor produced in these cells is negligible.

  3. Velocardiofacial syndrome in father and daughter: What is the mechanism for the deletion 22(q11.2q11.2) in only the daughter?

    Energy Technology Data Exchange (ETDEWEB)

    Magenis, R.E.; Gunter, K.; Toth-Fejel, S. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others

    1994-09-01

    E.G. had marked feeding difficulty noted at birth; the cause was determined to be a paralyzed palate. In 1992 chromosome studies were performed because of the provisional diagnosis of velocardiofacial syndrome, and a small interstitial deletion of chromosome 22 was found. Recently the family was seen in our Genetics Clinic. The father had unusual facial features shared by his daughter, a paralyzed upper lip and a history of repaired Tetralogy of Fallot. His chromosomes appeared normal. FISH studies were performed on the child`s peripheral blood using the ONCOR DiGeorge region probe (D22S75) and the deletion verified. However, the father`s chromosomes were not deleted for the ONCOR probe (D22S75) and probe DO832 sent to us by Peter Scambler. Skin cells were then obtained and no deletion was detected in a total of 66 cells examined using both probes. Several questions arise from these data: does the father have velocardiofacial syndrome? Does he have occult mosaicism? Does he have a molecular deletion not detected by the probes used? And was this deletion somehow {open_quotes}amplified{close_quotes} in his daughter?

  4. Molecular and cellular pathways associated with chromosome 1p deletions during colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Payne CM

    2011-05-01

    Full Text Available Claire M Payne, Cheray Crowley-Skillicorn, Carol Bernstein, Hana Holubec, Harris BernsteinDepartment of Cell Biology and Anatomy, College of Medicine, University of Arizona Tucson, AZ, USAAbstract: Chromosomal instability is a major pathway of sporadic colon carcinogenesis. Chromosome arm 1p appears to be one of the “hot spots” in the non-neoplastic mucosa that, when deleted, is associated with the initiation of carcinogenesis. Chromosome arm 1p contains genes associated with DNA repair, spindle checkpoint function, apoptosis, multiple microRNAs, the Wnt signaling pathway, tumor suppression, antioxidant activities, and defense against environmental toxins. Loss of 1p is dangerous since it would likely contribute to genomic instability leading to tumorigenesis. The 1p deletion-associated colon carcinogenesis pathways are reviewed at the molecular and cellular levels. Sporadic colon cancer is strongly linked to a high-fat/low-vegetable/low-micronutrient, Western-style diet. We also consider how selected dietary-related compounds (eg, excess hydrophobic bile acids, and low levels of folic acid, niacin, plant-derived antioxidants, and other modulatory compounds might affect processes leading to chromosomal deletions, and to the molecular and cellular pathways specifically altered by chromosome 1p loss.Keywords: chromosome 1p, colon carcinogenesis, molecular pathways, cellular pathways

  5. Systematic Identification of Determinants for Single-Strand Annealing-Mediated Deletion Formation in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Maia Segura-Wang

    2017-10-01

    Full Text Available To ensure genomic integrity, living organisms have evolved diverse molecular processes for sensing and repairing damaged DNA. If improperly repaired, DNA damage can give rise to different types of mutations, an important class of which are genomic structural variants (SVs. In spite of their importance for phenotypic variation and genome evolution, potential contributors to SV formation in Saccharomyces cerevisiae (budding yeast, a highly tractable model organism, are not fully recognized. Here, we developed and applied a genome-wide assay to identify yeast gene knockout mutants associated with de novo deletion formation, in particular single-strand annealing (SSA-mediated deletion formation, in a systematic manner. In addition to genes previously linked to genome instability, our approach implicates novel genes involved in chromatin remodeling and meiosis in affecting the rate of SSA-mediated deletion formation in the presence or absence of stress conditions induced by DNA-damaging agents. We closely examined two candidate genes, the chromatin remodeling gene IOC4 and the meiosis-related gene MSH4, which when knocked-out resulted in gene expression alterations affecting genes involved in cell division and chromosome organization, as well as DNA repair and recombination, respectively. Our high-throughput approach facilitates the systematic identification of processes linked to the formation of a major class of genetic variation.

  6. Juvenile Moyamoya and Craniosynostosis in a Child with Deletion 1p32p31: Expanding the Clinical Spectrum of 1p32p31 Deletion Syndrome and a Review of the Literature

    Directory of Open Access Journals (Sweden)

    Paolo Prontera

    2017-09-01

    Full Text Available Moyamoya angiopathy (MA is a rare cerebrovascular disorder characterised by the progressive occlusion of the internal carotid artery. Its aetiology is uncertain, but a genetic background seems likely, given the high MA familial rate. To investigate the aetiology of craniosynostosis and juvenile moyamoya in a 14-year-old male patient, we performed an array-comparative genomic hybridisation revealing a de novo interstitial deletion of 8.5 Mb in chromosome region 1p32p31. The deletion involved 34 protein coding genes, including NF1A, whose haploinsufficiency is indicated as being mainly responsible for the 1p32-p31 chromosome deletion syndrome phenotype (OMIM 613735. Our patient also has a deleted FOXD3 of the FOX gene family of transcription factors, which plays an important role in neural crest cell growth and differentiation. As the murine FOXD3−/− model shows craniofacial anomalies and abnormal common carotid artery morphology, it can be hypothesised that FOXD3 is involved in the pathogenesis of the craniofacial and vascular defects observed in our patient. In support of our assumption, we found in the literature another patient with a syndromic form of MA who had a deletion involving another FOX gene (FOXC1. In addition to describing the clinical history of our patient, we have reviewed all of the available literature concerning other patients with a 1p32p31 deletion, including cases from the Decipher database, and we have also reviewed the genetic disorders associated with MA, which is a useful guide for the diagnosis of syndromic form of MA.

  7. Numt-mediated double-strand break repair mitigates deletions during primate genome evolution.

    Directory of Open Access Journals (Sweden)

    Einat Hazkani-Covo

    2008-10-01

    Full Text Available Non-homologous end joining (NHEJ is the major mechanism of double-strand break repair (DSBR in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs. Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler

  8. 41 CFR 51-2.3 - Notice of proposed addition or deletion.

    Science.gov (United States)

    2010-07-01

    ... addition or deletion. 51-2.3 Section 51-2.3 Public Contracts and Property Management Other Provisions... or deletion. At least 30 days prior to the Committee's consideration of the addition or deletion of a... Register announcing the proposed addition or deletion and providing interested persons an opportunity to...

  9. 10 CFR 9.19 - Segregation of exempt information and deletion of identifying details.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Segregation of exempt information and deletion of... Information Act Regulations § 9.19 Segregation of exempt information and deletion of identifying details. (a... deletions are made from parts of the record by computer, the amount of information deleted will be indicated...

  10. BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

    International Nuclear Information System (INIS)

    Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Sakaguchi, Nobuo

    2006-01-01

    BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM lo IgD hi B-cells but not in AICD susceptible immature IgM hi IgD lo B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23 hi B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack of G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells

  11. Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight

    Directory of Open Access Journals (Sweden)

    Corey Nislow

    2015-01-01

    Full Text Available Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

  12. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    Science.gov (United States)

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  13. Studies on the Nucleotide Sequence, Transcription and Deletion Analysis of the BmNPV Protein Kinase Gene.

    Science.gov (United States)

    Zhang, Chuan-Xi; Hu, Cui; Wu, Xiang-Fu

    1998-01-01

    The coding region of BmvPK-1 gene of Bombyx mori NPV (Strain ZJ8) is 828 nt long and encodes a 276 aa polypeptide with predicted molecular mass of 32 kD. Dot blot analysis showed its mRNA to be gene is first detectable at 18 h p.i. and reaching the highest transcriptional level at 48 h p.i. The result suggested that BmvPK-1 gene is a late or very late gene. The most conserved 365 bp of the BmvPK-1 gene was deleted in a transfer vector (pUCPK-lac), and a report gene (lacZ) was inserted in the deleted position. Cotransfection of BmN cells with pUCPK-lac DNA and BmNPV DNA resulted in the recombinant virus which expressed detectable product of lacZ gene. But the virus with the deleted BmvPK-1 gene could not be isolated from the wild BmNPV by plaque purification method. The result showed that the BmvPK-1 gene deleted virus can multiply only with the help of the product of this gene from the wild type virus, and the gene is necessary for the virus to finish its life cycle in the cultured cells.

  14. De novo interstitial deletions of 9q22.1-22.3 in two unrelated cases with different phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Mohamed, A.N.; Bawle, E.; Conard, J. [Wayne State Univ., Detroit, MI (United States)] [and others

    1994-09-01

    Deletions involving the long arm of chromosome 9 are rare. A recent review, particularly with deletions of 9q22-32 region, failed to recognize a distinct pattern of dysmorphies and malformations. Herein, we described two phenotypically abnormal unrelated cases with interstitial deletion of chromosome 9 at band q22.1-q22.3. Parents of both cases exhibited normal karyotypes, indicating that the deletions were de novo events. Therefore, the clinical features present in these two cases can be attributed to partial monosomy for the deleted band 9q22. The first case was a 2-day-old baby with ambiguous genitalia, hydrocephalus, cleft palate and lip, polycystic kidney, absence of uterus on ultrasound and one gonad in the labiosacral region. Chromosome analysis showed a male karyotype, 46,XY,del(9)(q22.1q22.3). The absence of monosomy X cell line and the normal histology of testicular tissue were against the diagnosis of mixed gonadal dysgenesis or XY gonadal dysgenesis. The second 3-day-old newborn baby girl presented with right side hypoplastic heart and pulmonary atresia. In addition, the patient showed multiple dysmorphic features including epicanthal fold, low-set ears, depressed nasal bridge, hypertelorism, and micrognathia. The uvula is absent with slight cleft palate. Bilateral clinodactyly of 5th fingers and severe club feet were also present. The external genitalia was of a normal female phenotype. Chromosome study also indicated interstatial deletion of band 9q22. Although both cases appeared to have the same chromosomal anomalies, neither a discrete facial appearance nor a common pattern of malformations was noted.

  15. Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

    Science.gov (United States)

    Kostner, David; Rachinger, Michael; Liebl, Wolfgang; Ehrenreich, Armin

    2017-11-01

    Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

  16. Lynch syndrome-associated extracolonic tumors are rare in two extended families with the same EPCAM deletion.

    Science.gov (United States)

    Lynch, Henry T; Riegert-Johnson, Douglas L; Snyder, Carrie; Lynch, Jane F; Hagenkord, Jill; Boland, C Richard; Rhees, Jennifer; Thibodeau, Stephen N; Boardman, Lisa A; Davies, Janine; Kuiper, Roland P; Hoogerbrugge, Nicoline; Ligtenberg, Marjolijn J L

    2011-10-01

    The Lynch syndrome (LS) is an inherited cancer syndrome showing a preponderance of colorectal cancer (CRC) in context with endometrial cancer and several other extracolonic cancers, which is due to pathogenic mutations in the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Some families were found to show a LS phenotype without an identified MMR mutation, although there was microsatellite instability and absence of MSH2 expression by immunohistochemistry. Studies of a subset of these families found a deletion at the 3' end of the epithelial cell adhesion molecule (EPCAM) gene, causing transcription read-through resulting in silencing of MSH2 through hypermethylation of its promoter. The tumor spectrum of such families appears to differ from classical LS. Our study of two large families (USA Family R and Dutch Family A) with an EPCAM deletion was carried out using each institution's standard family study protocol. DNA was extracted from peripheral blood and EPCAM deletion analysis was performed. Both families were found to harbor the same deletion at the 3' end of EPCAM. Analysis showed that the deletion originated from a common ancestor. Family R and Family A members showed segregation of CRC with the presence of this EPCAM mutation. Compared with classic LS, there were almost no extracolonic cancers. Members of Family R and Family A, all with the same EPCAM deletion, predominantly presented with CRC but no LS-associated endometrial cancer, confirming findings seen in other, smaller, LS families with EPCAM mutations. In these EPCAM mutation carriers, cancer surveillance should be focused on CRC.

  17. Protein kinase Cα deletion causes hypotension and decreased vascular contractility.

    Science.gov (United States)

    Wynne, Brandi M; McCarthy, Cameron G; Szasz, Theodora; Molina, Patrick A; Chapman, Arlene B; Webb, R Clinton; Klein, Janet D; Hoover, Robert S

    2018-03-01

    Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

  18. Fungal ABC Transporter Deletion and Localization Analysis

    NARCIS (Netherlands)

    Kovalchuk, A.; Weber, S.S.; Nijland, J.G.; Bovenberg, R.A.L.; Driessen, A.J.M.

    2012-01-01

    Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological

  19. Amino-acid composition after loop deletion drives domain swapping.

    Science.gov (United States)

    Nandwani, Neha; Surana, Parag; Udgaonkar, Jayant B; Das, Ranabir; Gosavi, Shachi

    2017-10-01

    Rational engineering of a protein to enable domain swapping requires an understanding of the sequence, structural and energetic factors that favor the domain-swapped oligomer over the monomer. While it is known that the deletion of loops between β-strands can promote domain swapping, the spliced sequence at the position of the loop deletion is thought to have a minimal role to play in such domain swapping. Here, two loop-deletion mutants of the non-domain-swapping protein monellin, frame-shifted by a single residue, were designed. Although the spliced sequence in the two mutants differed by only one residue at the site of the deletion, only one of them (YEIKG) promoted domain swapping. The mutant containing the spliced sequence YENKG was entirely monomeric. This new understanding that the domain swapping propensity after loop deletion may depend critically on the chemical composition of the shortened loop will facilitate the rational design of domain swapping. © 2017 The Protein Society.

  20. The Yeast Deletion Collection: A Decade of Functional Genomics

    Science.gov (United States)

    Giaever, Guri; Nislow, Corey

    2014-01-01

    The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. PMID:24939991

  1. Molecular studies of deletions at the human steroid sulfatase locus

    International Nuclear Information System (INIS)

    Shapiro, L.J.; Yen, P.; Pomerantz, D.; Martin, E.; Rolewic, L.; Mohandas, T.

    1989-01-01

    The human steroid sulfatase gene (STS) is located on the distal X chromosome short arm close to the pseudoautosomal region but in a segment of DNA that is unique to the X chromosome. In contrast to most X chromosome-encoded genes, STS expression is not extinguished during the process of X chromosome inactivation. Deficiency of STS activity produced the syndrome of X chromosome-linked ichthyosis, which is one of the most common inborn errors of metabolism in man. Approximately 90% of STS - individuals have large deletions at the STS locus. The authors and others have found that the end points of such deletions are heterogeneous in their location. One recently ascertained subject was observed to have a 40-kilobase deletion that is entirely intragenic, permitting the cloning and sequencing of the deletion junction. Studies of this patient and of other X chromosome sequences in other subjects permit some insight into the mechanism(s) responsible for generating frequent deletions on the short arm of the X chromosome

  2. Sorting genomes by reciprocal translocations, insertions, and deletions.

    Science.gov (United States)

    Qi, Xingqin; Li, Guojun; Li, Shuguang; Xu, Ying

    2010-01-01

    The problem of sorting by reciprocal translocations (abbreviated as SBT) arises from the field of comparative genomics, which is to find a shortest sequence of reciprocal translocations that transforms one genome Pi into another genome Gamma, with the restriction that Pi and Gamma contain the same genes. SBT has been proved to be polynomial-time solvable, and several polynomial algorithms have been developed. In this paper, we show how to extend Bergeron's SBT algorithm to include insertions and deletions, allowing to compare genomes containing different genes. In particular, if the gene set of Pi is a subset (or superset, respectively) of the gene set of Gamma, we present an approximation algorithm for transforming Pi into Gamma by reciprocal translocations and deletions (insertions, respectively), providing a sorting sequence with length at most OPT + 2, where OPT is the minimum number of translocations and deletions (insertions, respectively) needed to transform Pi into Gamma; if Pi and Gamma have different genes but not containing each other, we give a heuristic to transform Pi into Gamma by a shortest sequence of reciprocal translocations, insertions, and deletions, with bounds for the length of the sorting sequence it outputs. At a conceptual level, there is some similarity between our algorithm and the algorithm developed by El Mabrouk which is used to sort two chromosomes with different gene contents by reversals, insertions, and deletions.

  3. Deleting the Arntl clock gene in the granular layer of the mouse cerebellum

    DEFF Research Database (Denmark)

    Bering, Tenna; Carstensen, Mikkel Bloss; Rath, Martin Fredensborg

    2017-01-01

    nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable. To investigate the hypothesis that the circadian oscillator of the cerebellum...... is involved in circadian physiology and food anticipation, we therefore by use of Cre-LoxP technology generated a conditional knockout mouse with the core clock gene Arntl deleted specifically in granule cells of the cerebellum, since expression of clock genes in the cerebellar cortex is mainly located...

  4. MET gene exon 14 deletion created using the CRISPR/Cas9 system enhances cellular growth and sensitivity to a MET inhibitor.

    Science.gov (United States)

    Togashi, Yosuke; Mizuuchi, Hiroshi; Tomida, Shuta; Terashima, Masato; Hayashi, Hidetoshi; Nishio, Kazuto; Mitsudomi, Tetsuya

    2015-12-01

    MET splice site mutations resulting in an exon 14 deletion have been reported to be present in about 3% of all lung adenocarcinomas. Patients with lung adenocarcinoma and a MET splice site mutation who have responded to MET inhibitors have been reported. The CRISPR/Cas9 system is a recently developed genome-engineering tool that can easily and rapidly cause small insertions or deletions. We created an in vitro model for MET exon 14 deletion using the CRISPR/Cas9 system and the HEK293 cell line. The phenotype, which included MET inhibitor sensitivity, was then investigated in vitro. Additionally, MET splice site mutations were analyzed in several cancers included in The Cancer Genome Atlas (TCGA) dataset. An HEK293 cell line with a MET exon 14 deletion was easily and rapidly created; this cell line had a higher MET protein expression level, enhanced MET phosphorylation, and prolonged MET activation. In addition, a direct comparison of phenotypes using this system demonstrated enhanced cellular growth, colony formation, and MET inhibitor sensitivity. In the TCGA dataset, lung adenocarcinomas had the highest incidence of MET exon 14 deletions, while other cancers rarely carried such mutations. Approximately 10% of the lung adenocarcinoma samples without any of driver gene alterations carried the MET exon 14 deletion. These findings suggested that this system may be useful for experiments requiring the creation of specific mutations, and the present experimental findings encourage the development of MET-targeted therapy against lung cancer carrying the MET exon 14 deletion. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

    Science.gov (United States)

    Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

    2017-09-15

    Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

  6. Deletion of the pH sensor GPR4 decreases renal acid excretion.

    Science.gov (United States)

    Sun, Xuming; Yang, Li V; Tiegs, Brian C; Arend, Lois J; McGraw, Dennis W; Penn, Raymond B; Petrovic, Snezana

    2010-10-01

    Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

  7. Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors.

    Science.gov (United States)

    Catón, Javier; Luder, Hans-Ulrich; Zoupa, Maria; Bradman, Matthew; Bluteau, Gilles; Tucker, Abigail S; Klein, Ophir; Mitsiadis, Thimios A

    2009-04-15

    TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1-/- mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1-/- mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.

  8. A local-world node deleting evolving network model

    International Nuclear Information System (INIS)

    Gu Yuying; Sun Jitao

    2008-01-01

    A new type network growth rule which comprises node addition with the concept of local-world connectivity and node deleting is studied. A series of theoretical analysis and numerical simulation to the LWD network are conducted in this Letter. Firstly, the degree distribution p(k) of this network changes no longer pure scale free but truncates by an exponential tail and the truncation in p(k) increases as p a decreases. Secondly, the connectivity is tighter, as the local-world size M increases. Thirdly, the average path length L increases and the clustering coefficient decreases as generally node deleting increases. Finally, trends up when the local-world size M increases, so as to k max . Hence, the expanding local-world can compensate the infection of the node deleting

  9. A local-world node deleting evolving network model

    Energy Technology Data Exchange (ETDEWEB)

    Gu Yuying [Department of Mathematics, Tongji University, Shanghai 200092 (China); Sun Jitao [Department of Mathematics, Tongji University, Shanghai 200092 (China)], E-mail: sunjt@sh163.net

    2008-06-16

    A new type network growth rule which comprises node addition with the concept of local-world connectivity and node deleting is studied. A series of theoretical analysis and numerical simulation to the LWD network are conducted in this Letter. Firstly, the degree distribution p(k) of this network changes no longer pure scale free but truncates by an exponential tail and the truncation in p(k) increases as p{sub a} decreases. Secondly, the connectivity is tighter, as the local-world size M increases. Thirdly, the average path length L increases and the clustering coefficient decreases as generally node deleting increases. Finally, trends up when the local-world size M increases, so as to k{sub max}. Hence, the expanding local-world can compensate the infection of the node deleting.

  10. Monoamine oxidase deficiency in males with an X chromosome deletion.

    Science.gov (United States)

    Sims, K B; de la Chapelle, A; Norio, R; Sankila, E M; Hsu, Y P; Rinehart, W B; Corey, T J; Ozelius, L; Powell, J F; Bruns, G

    1989-01-01

    Mapping of the human MAOA gene to chromosomal region Xp21-p11 prompted our study of two affected males in a family previously reported to have Norrie disease resulting from a submicroscopic deletion in this chromosomal region. In this investigation we demonstrate in these cousins deletion of the MAOA gene, undetectable levels of MAO-A and MAO-B activities in their fibroblasts and platelets, respectively, loss of mRNA for MAO-A in fibroblasts, and substantial alterations in urinary catecholamine metabolites. The present study documents that a marked deficiency of MAO activity is compatible with life and that genes for MAO-A and MAO-B are near each other in this Xp chromosomal region. Some of the clinical features of these MAO deletion patients may help to identify X-linked MAO deficiency diseases in humans.

  11. The diagnosis and molecular analysis of a novel 21.9kb deletion (Qinzhou type deletion) causing α+ thalassemia.

    Science.gov (United States)

    Long, Ju; Yan, Shanhuo; Lao, Kegan; Pang, Wanrong; Ye, Xuehe; Sun, Lei

    2014-04-01

    α-Thalassemia is a common single-gene genetic disease that can cause Hb Bart's hydrops fetalis and Hb H disease in tropical and subtropical regions. When examining conventional thalassemia genes, an only detected --(SEA) genotype sample needs further analysis. In doing so, we found a novel 21.9kb deletion (Qinzhou type deletion). The deletion position of the novel 21.9kb deletion is from 14373bp to 36299bp of the α-globin gene cluster (NG_000006.1); thus, there exists a 21927bp sequence deletion, into which a 29bp sequence is added. After sequence analysis, a group of Gap-PCR primers were synthesized to diagnose this novel thalassemia genotype. Through pedigree analysis, we deduced that the propositus obtained the novel alleles from her mother. The genotype of this propositus is --(SEA)/-α(21.9) and its phenotype conforms to the characteristics of Hb H disease, establishing that the combination between -α(21.9) genotype and α(0) genotype can lead to Hb H disease. By molecular analysis, we established that this case fits the characteristic of an α(+) thalassemia genotype. © 2013.

  12. Association between BIM deletion polymorphism and clinical outcome of EGFR-mutated NSCLC patient with EGFR-TKI therapy: A meta-analysis.

    Science.gov (United States)

    Ma, Ji-Yong; Yan, Hai-Jun; Gu, Wei

    2015-01-01

    BIM deletion polymorphism was deemed to be associated with downregulation of BIM, resulting in a decreased apoptosis induced by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutation-positive non-small cell lung cancer (NSCLC). However, accumulating evidences concerning the association between BIM deletion polymorphism and efficacy of EGFR-TKI and survival in EGFR-mutation-driven NSCLC patient reported contradictory results. A meta-analysis was conducted by combing six original eligible studies including 871 NSCLC patients. Our study showed that BIM deletion polymorphism was significantly associated with poor response to EGFR-TKI therapy in mutant EGFRNSCLC patients (P(h) = 0.309, P(z) = 0.001, OR = 0.39, 95% confidence interval (CI) = 0.23-0.67). Disease control rate (DCR) in mutant EGFRNSCLC patient with treatment of EGFR-TKI was significantly decreased in patients with BIM deletion polymorphism comparing to patients harbored BIM wild variant (P(h) = 0.583, P(Z) = 0.007, OR = 0.46, 95%CI = 0.25-0.85). EGFR mutation-derived NSCLC patient carrying BIM deletion polymorphism had a shorter progression-free survival (PFS; P(h) deletion polymorphism might be a cause that contributes to primary EGFR-TKI resistance, and it could be used as a genetic predictor for EGFR-TKI outcome and an independent prognostic factor of EGFR mutation-driven NSCLC patient.

  13. Pseudotumor of the pituitary due to PROP-1 deletion.

    Science.gov (United States)

    Teinturier, C; Vallette, S; Adamsbaum, C; Bendaoud, M; Brue, T; Bougnères, P F

    2002-01-01

    Hypopituitarism associated with pituitary mass in childhood is most frequently the consequence of craniopharyngioma or Rathke's cleft cyst. We report a patient with an intrasellar pseudotumor associated with hypopituitarism, which led us to a misdiagnosis of intrasellar craniopharyngioma. After spontaneous involution of the mass, diagnosis was revised. DNA analysis showed a deletion in the Prophet of Pit-1 (PROP-1) gene, a pituitary transcription factor. It is important to recognize that a PROP-1 deletion can cause pituitary pseudotumor that can be mistaken for a craniopharyngioma or Rathke's pouch cyst.

  14. Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors

    Directory of Open Access Journals (Sweden)

    Kawe Martin

    2009-01-01

    Full Text Available Abstract Background Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. Results We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. Conclusion The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

  15. Grin1 receptor deletion within CRF neurons enhances fear memory.

    Directory of Open Access Journals (Sweden)

    Georgette Gafford

    Full Text Available Corticotropin releasing factor (CRF dysregulation is implicated in mood and anxiety disorders such as posttraumatic stress disorder (PTSD. CRF is expressed in areas engaged in fear and anxiety processing including the central amygdala (CeA. Complicating our ability to study the contribution of CRF-containing neurons to fear and anxiety behavior is the wide variety of cell types in which CRF is expressed. To manipulate specific subpopulations of CRF containing neurons, our lab has developed a mouse with a Cre recombinase gene driven by a CRF promoter (CRFp3.0Cre (Martin et al., 2010. In these studies, mice that have the gene that encodes NR1 (Grin1 flanked by loxP sites (floxed were crossed with our previously developed CRFp3.0Cre mouse to selectively disrupt Grin1 within CRF containing neurons (Cre+/fGrin1+. We find that disruption of Grin1 in CRF neurons did not affect baseline levels of anxiety, locomotion, pain sensitivity or exploration of a novel object. However, baseline expression of Grin1 was decreased in Cre+/fGrin1+ mice as measured by RTPCR. Cre+/fGrin1+ mice showed enhanced auditory fear acquisition and retention without showing any significant effect on fear extinction. We measured Gria1, the gene that encodes AMPAR1 and the CREB activator Creb1 in the amygdala of Cre+/fGrin1+ mice after fear conditioning. Both Gria1 and Creb1 were enhanced in the amygdala after training. To determine if the Grin1-expressing CRF neurons within the CeA are responsible for the enhancement of fear memory in adults, we infused a lentivirus with Cre driven by a CRF promoter (LV pCRF-Cre/fGrin1+ into the CeA of floxed Grin1 mice. Cre driven deletion of Grin1 specifically within CRF expressing cells in the CeA also resulted in enhanced fear memory acquisition and retention. Altogether, these findings suggest that selective disruption of Grin1 within CeA CRF neurons strongly enhances fear memory.

  16. Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Rinker, Torri E.; Baker, Scott E.

    2007-01-29

    Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In this pelleted form, A. niger has increased citric acid production as compared to growth in filamentous form. Chitin is a crucial component of the cell wall of filamentous fungi. Alterations in the deposition or production of chitin may have profound effects on the morphology of the organism. In order to study the role of chitin synthesis in pellet formation we have deleted a chitin synthase gene (csmA) in Aspergillus niger strain ATCC 11414 using a PCR based deletion construct. This class of chitin synthases is only found in filamentous fungi and is not present in yeasts. The csmA genes contain a myosin motor domain at the N-terminus and a chitin synthesis domain at the C-terminus. They are believed to contribute to the specialized polar growth observed in filamentous fungi that is lacking in yeasts. The csmA deletion strain (csmAΔ) was subjected to minimal media with and without osmotic stabilizers as well as tested in citric acid production media. Without osmotic stabilizers, the mutant germlings were abnormally swollen, primarily in the subapical regions, and contained large vacuoles. However, this swelling is ultimately not inhibitory to growth as the germlings are able to recover and undergo polar growth. Colony formation was largely unaffected in the absence of osmotic stabilizers. In citric acid production media csmAΔ was observed to have a 2.5 fold increase in citric acid production. The controlled expression of this class of chitin synthases may be useful for improving production of organic acids in filamentous fungi.

  17. Catalase deletion promotes prediabetic phenotype in mice.

    Science.gov (United States)

    Heit, Claire; Marshall, Stephanie; Singh, Surrendra; Yu, Xiaoqing; Charkoftaki, Georgia; Zhao, Hongyu; Orlicky, David J; Fritz, Kristofer S; Thompson, David C; Vasiliou, Vasilis

    2017-02-01

    Hydrogen peroxide is produced endogenously and can be toxic to living organisms by inducing oxidative stress and cell damage. However, it has also been identified as a signal transduction molecule. By metabolizing hydrogen peroxide, catalase protects cells and tissues against oxidative damage and may also influence signal transduction mechanisms. Studies suggest that acatalasemic individuals (i.e., those with very low catalase activity) have a higher risk for the development of diabetes. We now report catalase knockout (Cat -/- ) mice, when fed a normal (6.5% lipid) chow, exhibit an obese phenotype that manifests as an increase in body weight that becomes more pronounced with age. The mice demonstrate altered hepatic and muscle lipid deposition, as well as increases in serum and hepatic triglycerides (TGs), and increased hepatic transcription and protein expression of PPARγ. Liver morphology revealed steatosis with inflammation. Cat -/- mice also exhibited pancreatic morphological changes that correlated with impaired glucose tolerance and increased fasting serum insulin levels, conditions consistent with pre-diabetic status. RNA-seq analyses revealed a differential expression of pathways and genes in Cat -/- mice, many of which are related to metabolic syndrome, diabetes, and obesity, such as Pparg and Cidec. In conclusion, the results of the present study show mice devoid of catalase develop an obese, pre-diabetic phenotype and provide compelling evidence for catalase (or its products) being integral in metabolic regulation. Copyright © 2016. Published by Elsevier Inc.

  18. Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease.

    Science.gov (United States)

    Kulshrestha, R; Burton-Jones, S; Antoniadi, T; Rogers, M; Jaunmuktane, Z; Brandner, S; Kiely, N; Manuel, R; Willis, T

    2017-08-01

    X-linked Charcot-Marie-Tooth disease (CMT) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene. This gene has nerve specific P2 promoter that work synergistically with SOX10 and EGR2 genes to initiate transcription. Mutation in this region is known to cause Schwann cell dysfunction. A single large family of X linked peripheral neuropathy was identified in our practice. Next generation sequencing for targeted panel assay identified an upstream exon-splicing deletion identified extending from nucleotide c.-5413 to approximately - c.-49. This matches the sequence of 32 nucleotides at positions c.*218-*249 in the 3'UTR downstream of the GJB1 gene. The deleted fragment included the entire P2 promoter region. The deletion segregated with the disease. To our knowledge a deletion of the P2 promoter alone as a cause of CMT has not been reported previously. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

    Energy Technology Data Exchange (ETDEWEB)

    Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

  20. Immune Modulation of NYVAC-Based HIV Vaccines by Combined Deletion of Viral Genes that Act on Several Signalling Pathways

    Directory of Open Access Journals (Sweden)

    Carmen Elena Gómez

    2017-12-01

    Full Text Available An HIV-1 vaccine continues to be a major target to halt the AIDS pandemic. The limited efficacy of the RV144 phase III clinical trial with the canarypox virus-based vector ALVAC and a gp120 protein component led to the conclusion that improved immune responses to HIV antigens are needed for a more effective vaccine. In non-human primates, the New York vaccinia virus (NYVAC poxvirus vector has a broader immunogenicity profile than ALVAC and has been tested in clinical trials. We therefore analysed the HIV immune advantage of NYVAC after removing viral genes that act on several signalling pathways (Toll-like receptors—TLR—interferon, cytokines/chemokines, as well as genes of unknown immune function. We generated a series of NYVAC deletion mutants and studied immune behaviour (T and B cell to HIV antigens and to the NYVAC vector in mice. Our results showed that combined deletion of selected vaccinia virus (VACV genes is a valuable strategy for improving the immunogenicity of NYVAC-based vaccine candidates. These immune responses were differentially modulated, positive or negative, depending on the combination of gene deletions. The deletions also led to enhanced antigen- or vector-specific cellular and humoral responses. These findings will facilitate the development of optimal NYVAC-based vaccines for HIV and other diseases.

  1. Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

    Science.gov (United States)

    Toyota, S; Hirosawa, S; Aoki, N

    1994-02-01

    Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

  2. Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

    DEFF Research Database (Denmark)

    Honoré, Bent; Buus, Søren; Claësson, Mogens H

    2008-01-01

    ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensiona......ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two...... alpha type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin beta-3 chain, a 25 kDa actin fragment, proteasome subunit beta type 9, cofilin-1 and glia...

  3. Homozygous Deletions and Recurrent Amplifications Implicate New Genes Involved in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Wennuan Liu

    2008-08-01

    Full Text Available Prostate cancer cell lines provide ideal in vitro systems for the identification and analysis of prostate tumor suppressors and oncogenes. A detailed characterization of the architecture of prostate cancer cell line genomes would facilitate the study of precise roles of various genes in prostate tumorigenesis in general. To contribute to such a characterization, we used the GeneChip 500K single nucleotide polymorphic (SNP array for analysis of genotypes and relative DNA copy number changes across the genome of 11 cell lines derived from both normal and cancerous prostate tissues. For comparison purposes, we also examined the alterations observed in the cell lines in tumor/normal pairs of clinical samples from 72 patients. Along with genome-wide maps of DNA copy number changes and loss of heterozygosity for these cell lines, we report previously unreported homozygous deletions and recurrent amplifications in prostate cancers in this study. The homozygous deletions affected a number of biologically important genes, including PPP2R2A and BNIP3L identified in this study and CDKN2A/CDKN2B reported previously. Although most amplified genomic regions tended to be large, amplifications at 8q24.21 were of particular interest because the affected regions are relatively small, are found in multiple cell lines, are located near MYC, an oncogene strongly implicated in prostate tumorigenesis, and are known to harbor SNPs that are associated with inherited susceptibility for prostate cancer. The genomic alterations revealed in this study provide an important catalog of positional information relevant to efforts aimed at deciphering the molecular genetic basis of prostate cancer.

  4. Deletion Mutagenesis and Identification of Causative Mutations in Maize.

    Science.gov (United States)

    Jia, Shangang; Li, Aixia; Zhang, Chi; Holding, David

    2018-01-01

    We describe a method for gamma-irradiation of mature maize seeds to generate mutants with opaque endosperm and reduced kernel fill phenotypes. We also describe methods for mapping mutants and identifying causal gene mutations. Using this method, a population of 1788M2 families and 47 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes was developed. For molecular characterization of the mutants, we utilized a novel functional genomics platform that combines separate Bulked Segregant RNA and exome sequencing data sets (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. We also describe the use of exome capture sequencing of F2 mutant and normal pools to perform mapping and candidate gene identification without the need for separate RNA-seq (BSEx-seq). To exemplify the utility of the deletion mutants for functional genomics and provide proof-of-concept for the bioinformatics platform, we summarize the identification of the causative deletion in two mutants. Mutant 937, which was characterized by BSREx-seq, harbors a 6203-bp in-frame deletion covering six exons within the Opaque-1 gene on chromosome 4. Preliminary investigation of opaque mutant 1486 with BSEx-seq shows a tight mapping interval and associated deletion on chromosome 10.

  5. Genetics Home Reference: 22q13.3 deletion syndrome

    Science.gov (United States)

    ... 5 links) Diagnostic Tests Drug Therapy Genetic Counseling Palliative Care Surgery and Rehabilitation Related Information How are genetic ... Veltman JA, de Vries BB. Molecular characterisation of patients with subtelomeric 22q ... L, Enns GM, Hoyme HE. Terminal 22q deletion syndrome: a newly recognized cause of ...

  6. 78 FR 21348 - Procurement List; Additions and Deletions; Recissions

    Science.gov (United States)

    2013-04-10

    ... COMMITTEE FOR PURCHASE FROM PEOPLE WHO ARE BLIND OR SEVERELY DISABLED Procurement List; Additions and Deletions; Recissions AGENCY: Committee for Purchase From People Who Are Blind or Severely Disabled. ACTION: Rescission of Previous Procurement List Decision. SUMMARY: The Committee for Purchase...

  7. Genetics Home Reference: 2q37 deletion syndrome

    Science.gov (United States)

    ... on PubMed or Free article on PubMed Central Casas KA, Mononen TK, Mikail CN, Hassed SJ, Li S, ... 2005 Aug 18. Citation on PubMed Falk RE, Casas KA. Chromosome 2q37 deletion: clinical and molecular aspects. ...

  8. The detection of large deletions or duplications in genomic DNA.

    Science.gov (United States)

    Armour, J A L; Barton, D E; Cockburn, D J; Taylor, G R

    2002-11-01

    While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications. Copyright 2002 Wiley-Liss, Inc.

  9. 78 FR 24733 - Procurement List, Additions and Deletions

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    2013-04-26

    ... Purchase From People Who Are Blind or Severely Disabled published notices of proposed additions to the... Services Administration. Portable Desktop Clipboard, 9\\1/2\\ W x 1\\1/2\\ D x 13\\1/2\\ H NSN: 7510-00-NIB-2133... for Purchase From People Who Are Blind or Severely Disabled published notices of proposed deletions...

  10. 76 FR 35415 - Procurement List; Proposed Additions and Deletions

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    2011-06-17

    ... Command, Natick, MA. SERVICE: Service Type/Location: Laundry Services, Department of Veterans Affairs... proposing to add products and a service to the Procurement List that will be furnished by nonprofit agencies employing persons who are blind or have other severe disabilities, and deletes products and a service...

  11. 76 FR 14942 - Procurement List; Additions and Deletions

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    2011-03-18

    ... DFAC. Service Type/Location: Laundry & Dry Cleaning Service, F.E. Warren, AFB, WY. NPA: Goodwill... Service Type/Location: Laundry Service, Atlanta VA Medical Center, Decatur, GA. NPA: GINFL Services, Inc...: Additions to and deletions from the Procurement List. SUMMARY: This action adds services to the Procurement...

  12. 75 FR 41449 - Procurement List Additions and Deletion

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    2010-07-16

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  13. 77 FR 31335 - Procurement List; Proposed Additions and Deletion

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    2012-05-25

    .... Services Service Type/Location: Laundry and Dry Cleaning Service, Buckley Air Force Base Lodging & Medical... products and services to the Procurement List that will be furnished by nonprofit agencies employing persons who are blind or have other severe disabilities, and deletes a service previously provided by such...

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  15. 78 FR 17641 - Procurement List; Proposed Addition and Deletion

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  16. 78 FR 45183 - Procurement List; Proposed Addition and Deletions

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    2013-07-26

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  17. 78 FR 68823 - Procurement List Proposed Additions and Deletions

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    2013-11-15

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  18. 78 FR 32631 - Procurement List; Proposed Additions and Deletions

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  19. 78 FR 43180 - Procurement List; Proposed Additions and Deletions

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    2013-07-19

    ... for Purchase From People Who Are Blind or Severely Disabled, 1401 S. Clark Street, Suite 10800... COMMITTEE FOR PURCHASE FROM PEOPLE WHO ARE BLIND OR SEVERELY DISABLED Procurement List; Proposed Additions and Deletions AGENCY: Committee for Purchase from People Who Are Blind or Severely Disabled...

  20. Angiotensin-converting enzyme insertion/deletion gene ...

    Indian Academy of Sciences (India)

    Angiotensin-converting enzyme insertion/deletion gene polymorphism in cystic fibrosis patients. Sabrine Oueslati Sondess Hadj Fredj Hajer Siala Amina Bibi Hajer Aloulou Lamia Boughamoura Khadija Boussetta Sihem Barsaoui Taieb Messaoud. Research Note Volume 95 Issue 1 March 2016 pp 193-196 ...

  1. The insertion/deletion polymorphism of angiotensin-converting ...

    African Journals Online (AJOL)

    The association between type 2 diabetes mellitus (T2DM) and essential hypertension (EH) is not well understood. Both conditions result from an interaction of multiple genetic (ethnic) and environmental (geographical) factors. One possible genetic determinant is the angiotensin-converting enzyme (ACE) insertion/deletion ...

  2. Remarks on Causative Verbs and Object Deletion in English

    Science.gov (United States)

    Onozuka, Hiromi

    2007-01-01

    Rappaport Hovav and Levin [Rappaport Hovav, M., Levin, B., 1998. "Building verb meanings." In: Butt, M., Geuder, W. (Eds.), "The Projection of Arguments: Lexical and Compositional Factors." CSLI Publications, Stanford, pp. 97-134] contend that result verbs disallow object deletion because of their lexical semantic properties. Their point is that…

  3. [An updated review of 1p36 deletion (monosomy) syndrome].

    Science.gov (United States)

    Bello, Sabina; Rodríguez-Moreno, Antonio

    The Monosomy 1p36 deletion syndrome is part of the group of diseases known as Rare Diseases. The objective of the present work is to review the characteristics of Monosomy 1p36 deletion syndrome. The monosomy 1p36 deletion syndrome phenotype includes: dysmorphic craniofacial features; large anterior fontanelle, unibrow, deep-set eyes, epicanthus, wide nasal root/bridge, mandible hypoplasia, abnormal location of the pinna, philtrum and pointed chin; neurological alterations: seizures and hydrocephalus (in some cases). Cerebral malformations: ventricular hypertrophy, increased subarachnoid space, morphological alterations of corpus callosum, cortical atrophy, delays in myelinisation, periventricular leukomalacia and periventricular heterotopia. These alterations produce intellectual disability and delays in motor growth, communication skills, language, social and adaptive behaviour. It is Hearing and vision impairments are also observed in subjects with this syndrome, as well as alterations of cardiac, endocrine and urinary systems and alterations at skin and skeletal level. Approximately 100 cases have been documented since 1981. This rare disease is the most common subtelomeric-micro-deletion syndrome. In situ hybridization with fluorescence (FISH) and array-comparative genomic hybridization (CGH-array) are at present the two best diagnostic techniques. There is currently no effective medical treatment for this disease. Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. 78 FR 73503 - Procurement List Additions and Deletions

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    2013-12-06

    ... by the General Services Administration. NSN: MR 376--Resealable Bags, Holiday, 6.5'' x 5.875''. NSN..., Holiday, 24PC. NPA: Winston-Salem Industries for the Blind, Inc., Winston-Salem, NC. Contracting Activity... disabilities, and deletes a product and services from the Procurement List previously furnished by such...

  5. Genetics Home Reference: distal 18q deletion syndrome

    Science.gov (United States)

    ... 18q deletion syndrome chromosome 18q monosomy chromosome 18q- syndrome De Grouchy syndrome del(18q) syndrome monosomy 18q Related Information How ... MS, Tienari PJ, Wirtavuori KO, Valanne LK. 18q-syndrome: brain MRI shows poor differentiation of gray and white matter on ... RL, Hale DE, Rose SR, Leach RJ, Cody JD. The spectrum ...

  6. Construction of a psb C deletion strain in Synechocystis 6803.

    Science.gov (United States)

    Goldfarb, N; Knoepfle, N; Putnam-Evans, C

    1997-01-01

    Synechocystis 6803 is a cyanobacterium that carries out-oxygenic photosynthesis. We are interested in the introduction of mutations in the large extrinsic loop region of the CP43 protein of Photosystem II (PSII). CP43 appears to be required for the stable assembly of the PSII complex and also appears to play a role in photosynthetic oxygen evolution. Deletion of short segments of the large extrinsic loop results in mutants incapable of evolving oxygen. Alterations in psbC, the gene encoding CP43, are introduced into Synechocystis 6803 by transformation and homologous recombination. Specifically, plasmid constructs bearing the site-directed mutations are introduced into a deletion strain where the portion of the gene encoding the area of mutation has been deleted and replaced by a gene conferring antibiotic resistance. We have constructed a deletion strain of Synechocystis appropriate for the introduction of mutations in the large extrinsic loop of CP43 and have used it successfully to produce site-directed mutants.

  7. 77 FR 60969 - Procurement List; Proposed Additions and Deletions

    Science.gov (United States)

    2012-10-05

    ...., Wichita, KS. Contracting Activity: Defense Logistics Agency Troop Support, Philadelphia, PA. Coverage: C-List for 100% of the requirement of the Department of Defense, as aggregated by the Defense Logistics...., Portsmouth, VA. Contracting Activity: Dept. of the Army, W071 Endist Kansas City, Kansas City, MO. Deletions...

  8. Differential radiosensitivity among B cell subpopulations

    International Nuclear Information System (INIS)

    Riggs, J.E.

    1988-01-01

    The selective radiosensitivity of sIgM >> sIgD marginal zone B cells is associated with the selective loss of B cell function. The simultaneous restoration of impaired function and recovery of these cells with time supports this premise. B cell recovery, delayed one week after irradiation, is in progress at two weeks, and virtually complete by three weeks. XID mice reveal similar recovery kinetics although there are fewer recovering cells and these bear reduced levels of Ia. This observation represents additional evidence that xid B cells are distinct from those of normal mice. The simultaneous loss, and concurrent recovery, of sIgM >> sIgD B cells and TI-2 responsiveness in irradiated mice suggests the existence of a unique B cell subpopulation possessing both phenotypes. Additional support for this hypothesis is provided by demonstrating that splenocytes, depleted of IgD + cells adoptively reconstitute this response in XID mice. The peritoneal B cell pool, which, compared to the spleen, consist of increased numbers of sIgM >> sIgD B cells, is shown to be a source of radiosensitive B cells that are TI-2 responsive. These observations represent additional evidence for an association between sIgM >> sIgD B cells and TI-2 responsiveness

  9. Atherosclerotic lesions and mitochondria DNA deletions in brain microvessels: implication in the pathogenesis of Alzheimer's disease.

    Science.gov (United States)

    Aliev, Gjumrakch; Gasimov, Eldar; Obrenovich, Mark E; Fischbach, Kathryn; Shenk, Justin C; Smith, Mark A; Perry, George

    2008-01-01

    The pathogenesis that is primarily responsible for Alzheimer's disease (AD) and cerebrovascular accidents (CVA) appears to involve chronic hypoperfusion. We studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD biopsy samples and two transgenic mouse models of AD, yeast artificial chromosome (YAC) and C57B6/SJL Tg (+), which overexpress human amyloid beta precursor protein (AbetaPP). In situ hybridization using probes for normal and 5 kb deleted human and mouse mitochondrial DNA (mtDNA) was performed along with immunocytochemistry using antibodies against the Abeta peptide processed from AbetaPP, 8-hydroxy-2'-guanosine (8OHG), and cytochrome c oxidase (COX). More amyloid deposition, oxidative stress markers as well as mitochondrial DNA deletions and structural abnormalities were present in the vascular walls of the human AD samples and the AbetaPP-YAC and C57B6/SJL Tg (+) transgenic mice compared to age-matched controls. Ultrastructural damage in perivascular cells highly correlated with endothelial lesions in all samples. Therefore, pharmacological interventions, directed at correcting the chronic hypoperfusion state, may change the natural course of the development of dementing neurodegeneration.

  10. Deletion mutant defines DQ beta variants with DR4 positive DQw3 positive haplotypes

    International Nuclear Information System (INIS)

    Nepom, B.S.; Kim, S.J.; Nepom, G.T.

    1986-01-01

    We describe the production of an HLA deletion mutation by radiation mutagenesis of a DR4- and DQw3-homozygous, Dw4- and Dw14-heterozygous cell line designed to analyze polymorphisms associated with DR4 and DQw3. Southern blot analysis confirms a deletion of class I and class II genes on one haplotype. Variation in DQ beta alleles associated with DQw3 was previously described by characteristic RFLP patterns for a DQ beta bene. One pattern, which correlated precisely with A-10-83 monoclonal antibody reactivity (TA10), defined an allele which we call DQ''3.1''. The mutant cell line has lost the polymorphic bands on Southern blots corresponding to the DQ''3.1'' allele, while the intact Dw14 haplotype retains the alternate allele at DQ beta which is DQw-3 positive. TA10-negative. These data demonstrate the segregation of two DQw3 positive DQ beta allelic variants, both associated with DR4, which can be distinguished on the basis of both RFLP and monoclonal antibody reactivity

  11. Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis

    Science.gov (United States)

    Xuan, Shouhong; Borok, Matthew J.; Decker, Kimberly J.; Battle, Michele A.; Duncan, Stephen A.; Hale, Michael A.; Macdonald, Raymond J.; Sussel, Lori

    2012-01-01

    Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis. PMID:23006325

  12. UNC5B receptor deletion exacerbates tissue injury in response to AKI.

    Science.gov (United States)

    Ranganathan, Punithavathi; Jayakumar, Calpurnia; Navankasattusas, Sutip; Li, Dean Y; Kim, Il-man; Ramesh, Ganesan

    2014-02-01

    Netrin-1 regulates cell survival and apoptosis by activation of its receptors, including UNC5B. However, the in vivo role of UNC5B in cell survival during cellular stress and tissue injury is unknown. We investigated the role of UNC5B in cell survival in response to stress using mice heterozygously expressing the UNC5B gene (UNC5B(-/flox)) and mice with targeted homozygous deletion of UNC5B in kidney epithelial cells (UNC5B(-/flox/GGT-cre)). Mice were subjected to two different models of organ injury: ischemia reperfusion injury of the kidney and cisplatin-induced nephrotoxicity. Both mouse models of UNC5B depletion had normal organ function and histology under basal conditions. After AKI, however, UNC5B(-/flox/GGT-cre) mice exhibited significantly worse renal function and damage, increased tubular apoptosis, enhanced p53 activation, and exacerbated inflammation compared with UNC5B(-/flox) and wild-type mice. shRNA-mediated suppression of UNC5B expression in cultured tubular epithelial cells exacerbated cisplatin-induced cell death in a p53-dependent manner and blunted Akt phosphorylation. Inhibition of PI3 kinase similarly exacerbated cisplatin-induced apoptosis; in contrast, overexpression of UNC5B reduced cisplatin-induced apoptosis in these cells. Taken together, these results show that the netrin-1 receptor UNC5B plays a critical role in cell survival and kidney injury through Akt-mediated inactivation of p53 in response to stress.

  13. Margem de segurança do meloxicam em cães: efeitos deletérios nas células sangüíneas e trato gastrintestinal Margin of safety of meloxicam in dogs: deleterious effects on blood cells and gastrointestinal tract

    Directory of Open Access Journals (Sweden)

    Marilac Maria Arnaldo Alencar

    2003-06-01

    Full Text Available Este trabalho investigou a margem de segurança do inibidor COX-2, meloxicam, em cães, enfocando seus efeitos deletérios nas células sangüíneas e no trato gastrintestinal. Para tanto, após uma avaliação clínico-laboratorial, os cães foram distribuídos nos seguintes grupos: I (placebo; n= 3, II (piroxicam; 1,0mg kg-1; n= 5, III (meloxicam; 0,2mg kg-1; n= 5, IV (meloxicam;1,0mg kg-1; n= 5 e V (meloxicam; 2,0mg kg-1; n= 5. Os fármacos foram usados, por via oral, por 16 dias. No 17º dia, repetiu-se o hemograma completo e, então, procedeu-se a eutanásia seguida pela necrópsia. No grupo I, não houve alterações dignas de nota. No grupo II, todos os cães apresentaram episódios moderados de vômito e diarréia. O perfil celular sangüíneo não foi significativamente modificado. Em dois cães, houve redução no hematócrito e na hemoglobina. Na necropsia, observaram-se focos hemorrágicos e lesões gastriduodenais moderadas. A análise microscópica revelou a presença de gastrite e enterite ulcerativa. No grupo III, quatro cães (80% apresentaram vômito e diarréia, sem alteração no perfil celular sangüíneo. Na análise macroscópica, observaram-se lesões brandas na mucosa gástrica e focos hemorrágicos no duodeno em quatro cães. Na histopatologia, observaram-se lesões sugestivas de discreta gastroenterite. No grupo IV, os cinco cães tiveram vômito e diarréia sanguinolenta. Quatro deles (80% apresentaram anemia (p This study investigated the margin of safety of the COX-2e inhibitor, meloxicam on blood cells and gastrointestinal tract of dogs. After a clinical and laboratorial examination, the dogs were distributed in the following groups: I (placebo; n=3, II (piroxicam: 1.0mg kg-1; n=5, III (meloxicam: 0.2mg kg-1; n=5, IV (meloxican: 1.0mg kg-1; n=5 and V (meloxican: 2.0mg kg-1; n=5. The drugs were given orally for 16 days. On the 17th day the complete hemogram was repeated and the euthanasia and necropsy were then

  14. One in Four Individuals of African-American Ancestry Harbors a 5.5kb Deletion at chromosome 11q13.1

    Science.gov (United States)

    Zainabadi, Kayvan; Jain, Anuja V.; Donovan, Frank X.; Elashoff, David; Rao, Nagesh P.; Murty, Vundavalli V.; Chandrasekharappa, Settara C.; Srivatsan, Eri S.

    2014-01-01

    Cloning and sequencing of 5.5kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines has revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5kb sequence in 494 normal lymphocyte samples showed heterozygous deletion in 28.3% of African- American ancestry samples but only in 4.8% of Caucasian samples (pdeletion occurs in 27% of YRI (Yoruba – West African) population but none in non-African populations. The HapMap analysis further identified strong linkage disequilibrium between 5 single nucleotide polymorphisms and the 5.5kb deletion in the people of African ancestry. Computational analysis of 175kb sequence surrounding the deletion site revealed enhanced flexibility, low thermodynamic stability, high repetitiveness, and stable stem-loop/hairpin secondary structures that are hallmarks of common fragile sites. PMID:24412158

  15. Deletion affecting band 7q36 not associated with holoprosencephaly

    Energy Technology Data Exchange (ETDEWEB)

    Ebrahim, S.A.D.; Krivchenia, E.; Mohamed, A.N. [Wayne State Univ., Detroit, MI (United States)] [and others

    1994-09-01

    Although the appearance of 7q36 aberrations have been postulated to be responsible for holoprosencephaly (HPE), the presence of a de novo 7q36 deletion in fetus without HPE has not been reported. We report the first case of a fetus with 7q36 deletion but lacking HPE. Ultrasound examination of a 25-year-old G3P1 Caucasian female showed small head circumference with microcephaly at 28 weeks. Decreased amniotic fluid volume, bilateral renal dilatation and abnormal facial features were also noted. Chromosome analysis after cordocentesis showed an abnormal female karyotype with a deletion involving the chromosome band 7q36, 46,XX,del(7)(q36). Chromosome studies on the biological parents were normal. In view of the chromosome finding and after extensive counseling, the couple elected to terminate the pregnancy. The chromosome findings were confirmed by fetal blood chromosome analysis at termination. Post-mortem examination confirmed dysmorphic features including a depressed nasal bridge and large flat ears with no lobules, but no cleft lip or palate was noted. Internal abnormalities included a bicuspid pulmonary valve and abnormally located lungs. The brain weighed 190g (249 {plus_minus} 64g expected) and had symmetric cerebral hemispheres without evidence of HPE or other gross or microscopic malformation, except focal cerebellar cortical dysplasia. In summary, our patient showed a deletion of the same chromosomal band implicated in HPE but lacked HPE. This finding indicates that 7q36 deletion may be seen in the absence of HPE and suggests that other genetic mechanisms may be responsible for HPE in this setting.

  16. Rare copy number deletions predict individual variation in intelligence.

    Directory of Open Access Journals (Sweden)

    Ronald A Yeo

    2011-01-01

    Full Text Available Phenotypic variation in human intellectual functioning shows substantial heritability, as demonstrated by a long history of behavior genetic studies. Many recent molecular genetic studies have attempted to uncover specific genetic variations responsible for this heritability, but identified effects capture little variance and have proven difficult to replicate. The present study, motivated an interest in "mutation load" emerging from evolutionary perspectives, examined the importance of the number of rare (or infrequent copy number variations (CNVs, and the total number of base pairs included in such deletions, for psychometric intelligence. Genetic data was collected using the Illumina 1MDuoBeadChip Array from a sample of 202 adult individuals with alcohol dependence, and a subset of these (N = 77 had been administered the Wechsler Abbreviated Scale of Intelligence (WASI. After removing CNV outliers, the impact of rare genetic deletions on psychometric intelligence was investigated in 74 individuals. The total length of the rare deletions significantly and negatively predicted intelligence (r = -.30, p = .01. As prior studies have indicated greater heritability in individuals with relatively higher parental socioeconomic status (SES, we also examined the impact of ethnicity (Anglo/White vs. Other, as a proxy measure of SES; these groups did not differ on any genetic variable. This categorical variable significantly moderated the effect of length of deletions on intelligence, with larger effects being noted in the Anglo/White group. Overall, these results suggest that rare deletions (between 5% and 1% population frequency or less adversely affect intellectual functioning, and that pleotropic effects might partly account for the association of intelligence with health and mental health status. Significant limitations of this research, including issues of generalizability and CNV measurement, are discussed.

  17. Rare Copy Number Deletions Predict Individual Variation in Intelligence

    Science.gov (United States)

    Yeo, Ronald A.; Gangestad, Steven W.; Liu, Jingyu; Calhoun, Vince D.; Hutchison, Kent E.

    2011-01-01

    Phenotypic variation in human intellectual functioning shows substantial heritability, as demonstrated by a long history of behavior genetic studies. Many recent molecular genetic studies have attempted to uncover specific genetic variations responsible for this heritability, but identified effects capture little variance and have proven difficult to replicate. The present study, motivated an interest in “mutation load” emerging from evolutionary perspectives, examined the importance of the number of rare (or infrequent) copy number variations (CNVs), and the total number of base pairs included in such deletions, for psychometric intelligence. Genetic data was collected using the Illumina 1MDuoBeadChip Array from a sample of 202 adult individuals with alcohol dependence, and a subset of these (N = 77) had been administered the Wechsler Abbreviated Scale of Intelligence (WASI). After removing CNV outliers, the impact of rare genetic deletions on psychometric intelligence was investigated in 74 individuals. The total length of the rare deletions significantly and negatively predicted intelligence (r = −.30, p = .01). As prior studies have indicated greater heritability in individuals with relatively higher parental socioeconomic status (SES), we also examined the impact of ethnicity (Anglo/White vs. Other), as a proxy measure of SES; these groups did not differ on any genetic variable. This categorical variable significantly moderated the effect of length of deletions on intelligence, with larger effects being noted in the Anglo/White group. Overall, these results suggest that rare deletions (between 5% and 1% population frequency or less) adversely affect intellectual functioning, and that pleotropic effects might partly account for the association of intelligence with health and mental health status. Significant limitations of this research, including issues of generalizability and CNV measurement, are discussed. PMID:21298096

  18. Structural and Functional Consequences of Chaperone Site Deletion in αA-Crystallin

    Science.gov (United States)

    Santhoshkumar, Puttur; Karmakar, Srabani; Sharma, Krishna K.

    2016-01-01

    The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70–88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70–76 and αAΔ70–88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70–88 was completely inactive as a chaperone, while αAΔ70–76 demonstrated a 40–50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70–88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45 °C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70–88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53 °C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70–88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70–88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins. PMID:27524665

  19. Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus.

    Science.gov (United States)

    Yang, Ying-Jie; Wang, Ye; Li, Zhi-Feng; Gong, Ya; Zhang, Peng; Hu, Wen-Chao; Sheng, Duo-Hong; Li, Yue-Zhong

    2017-08-16

    The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

  20. Proglucagon Promoter Cre-Mediated AMPK Deletion in Mice Increases Circulating GLP-1 Levels and Oral Glucose Tolerance.

    Directory of Open Access Journals (Sweden)

    Sophie R Sayers

    Full Text Available Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1 in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK in these cells.Loss of AMPK from proglucagon-expressing cells was achieved using a preproglucagon promoter-driven Cre (iGluCre to catalyse recombination of floxed alleles of AMPKα1 and α2. Oral and intraperitoneal glucose tolerance were measured using standard protocols. L-cell mass was measured by immunocytochemistry. Hormone and peptide levels were measured by electrochemical-based luminescence detection or radioimmunoassay.Recombination with iGluCre led to efficient deletion of AMPK from intestinal L- and pancreatic alpha-cells. In contrast to mice rendered null for LKB1 using the same strategy, mice deleted for AMPK displayed an increase (WT: 0.05 ± 0.01, KO: 0.09±0.02%, p<0.01 in L-cell mass and elevated plasma fasting (WT: 5.62 ± 0.800 pg/ml, KO: 14.5 ± 1.870, p<0.01 and fed (WT: 15.7 ± 1.48pg/ml, KO: 22.0 ± 6.62, p<0.01 GLP-1 levels. Oral, but not intraperitoneal, glucose tolerance was significantly improved by AMPK deletion, whilst insulin and glucagon levels were unchanged despite an increase in alpha to beta cell ratio (WT: 0.23 ± 0.02, KO: 0.33 ± 0.03, p<0.01.AMPK restricts L-cell growth and GLP-1 secretion to suppress glucose tolerance. Targeted inhibition of AMPK in L-cells may thus provide a new therapeutic strategy in some forms of type 2 diabetes.

  1. Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

    Energy Technology Data Exchange (ETDEWEB)

    Champagne, Devin P., E-mail: devin.champagne@uvm.edu; Shockett, Penny E., E-mail: pshockett@selu.edu

    2014-03-15

    Highlights: • Examines illegitimate V(D)J deletion junctions in Notch1 and Bcl11b. • Suggests little influence of deletions alone on clonal outgrowth in wild-type mice. • No age or sex biases in frequency, clonality, or junctional processing observed. • Contrasts with previous results at TCRβ and HPRT1 loci. • Deletions in Bcl11b may be tolerated more easily than those in Notch1. - Abstract: Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is

  2. Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

    Science.gov (United States)

    Cohrs, Randall J; Lee, Katherine S; Beach, Addilynn; Sanford, Bridget; Baird, Nicholas L; Como, Christina; Graybill, Chiharu; Jones, Dallas; Tekeste, Eden; Ballard, Mitchell; Chen, Xiaomi; Yalacki, David; Frietze, Seth; Jones, Kenneth; Lenac Rovis, Tihana; Jonjić, Stipan; Haas, Jürgen; Gilden, Don

    2017-10-15

    The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture. IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture. Copyright © 2017 American Society for Microbiology.

  3. Dopaminergic Neuron-Specific Deletion of p53 Gene Attenuates Methamphetamine Neurotoxicity.

    Science.gov (United States)

    Lu, Tao; Kim, Paul P; Greig, Nigel H; Luo, Yu

    2017-08-01

    p53 plays an essential role in the regulation of cell death in dopaminergic (DA) neurons and its activation has been implicated in the neurotoxic effects of methamphetamine (MA). However, how p53 mediates MA neurotoxicity remains largely unknown. In this study, we examined the effect of DA-specific p53 gene deletion in DAT-p53KO mice. Whereas in vivo MA binge exposure reduced locomotor activity in wild-type (WT) mice, this was significantly attenuated in DAT-p53KO mice and associated with significant differences in the levels of the p53 target genes BAX and p21 between WT and DAT-p53KO. Notably, DA-specific deletion of p53 provided protection of substantia nigra pars reticulata (SNpr) tyrosine hydroxylase (TH) positive fibers following binge MA, with DAT-p53KO mice having less decline of TH protein levels in striatum versus WT mice. Whereas DAT-p53KO mice demonstrated a consistently higher density of TH fibers in striatum compared to WT mice at 10 days after MA exposure, DA neuron counts within the substantia nigra pars compacta (SNpc) were similar. Finally, supportive of these results, administration of a p53-specific inhibitor (PFT-α) provided a similarly protective effect on MA binge-induced behavioral deficits. Neither DA specific p53 deletion nor p53 pharmacological inhibition affected hyperthermia induced by MA binge. These findings demonstrate a specific contribution of p53 activation in behavioral deficits and DA neuronal terminal loss by MA binge exposure.

  4. Restoration of half the normal dystrophin sequence in a double-deletion Duchenne muscular dystrophy family

    Energy Technology Data Exchange (ETDEWEB)

    Hoop, R.C.; Schwartz, L.S.; Hoffman, E.P. [Univ. of Pittsburgh School of Medicine, Pittsburgh, PA (United States); Russo, L.S. [Univ. of Florida, Jacksonville, FL (United States); Riconda, D.L. [Orlando Regional Medical Center, Orlando, FL (United States)

    1994-02-01

    Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions-one in the 5{prime}, proximal deletional hotspot region, and the other in the 3{prime}, more distal deletional hotspot region. The second propositus showed only the 5{prime} deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, the authors show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier`s dystrophin genes, giving rise to a son with a partially {open_quotes}repaired{close_quotes} gene retaining only the 5{prime} deletion. 20 refs., 4 figs.

  5. Global transcriptional response of Saccharomyces cerevisiae to the deletion of SDH3

    DEFF Research Database (Denmark)

    Cimini, Donatella; Patil, Kiran Raosaheb; Schiraldi, Chiara

    2009-01-01

    Background: Mitochondrial respiration is an important and widely conserved cellular function in eukaryotic cells. The succinate dehydrogenase complex (Sdhp) plays an important role in respiration as it connects the mitochondrial respiratory chain to the tricarboxylic acid (TCA) cycle where...... it catalyzes the oxidation of succinate to fumarate. Cellular response to the Sdhp dysfunction (i.e. impaired respiration) thus has important implications not only for biotechnological applications but also for understanding cellular physiology underlying metabolic diseases such as diabetes. We therefore...... conditions is very low, deletion of SDH3 resulted in significant changes in the expression of several genes involved in various cellular processes ranging from metabolism to the cell-cycle. By using various bioinformatics tools we explored the organization of these transcriptional changes in the metabolic...

  6. RIP3 attenuates the pancreatic damage induced by deletion of ATG7.

    Science.gov (United States)

    Zhou, Xiaodong; Xie, Li; Xia, Leizhou; Bergmann, Frank; Büchler, Markus W; Kroemer, Guido; Hackert, Thilo; Fortunato, Franco

    2017-07-13

    Invalidation of pancreatic autophagy entails pancreatic atrophy, endocrine and exocrine insufficiency and pancreatitis. The aim of this study was to investigate whether depletion of Rip3, which is involved in necroptotic signaling, may attenuate the pancreatic atrophy and pancreatitis resulting from autophagy inhibition. Autophagy and necroptosis signaling were evaluated in mice lacking expression of Rip3 in all organs and Atg7 in the pancreas. Acinar cell death, inflammation and fibrosis were evaluated by using of a compendium of immunofluorescence methods and immunoblots. Mice deficient for pancreatic Atg7 developed acute pancreatitis, which progressed to chronic pancreatitis. This phenotype reduces autophagy, increase apoptosis and necroptosis, inflammation and fibrosis, as well as premature death of the animals. Knockout of Rip3 exacerbated the apoptotic death of acinar cells, increased tissue damage, reduced macrophage infiltration and further accelerated the death of the mice with Atg7-deficient pancreas. The pancreatic degeneration induced by autophagy inhibition was exacerbated by Rip3 deletion.

  7. Sequence homology at the breakpoint and clinical phenotype of mitochondrial DNA deletion syndromes.

    Science.gov (United States)

    Sadikovic, Bekim; Wang, Jing; El-Hattab, Ayman W; Landsverk, Megan; Douglas, Ganka; Brundage, Ellen K; Craigen, William J; Schmitt, Eric S; Wong, Lee-Jun C

    2010-12-20

    Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement.

  8. Effects of Deletion of the Streptococcus pneumoniae Lipoprotein Diacylglyceryl Transferase Gene lgt on ABC Transporter Function and on Growth In Vivo.

    NARCIS (Netherlands)

    Chimalapati, S.; Cohen, J.M.; Camberlein, E.; Macdonald, N.; Durmort, C.; Vernet, T.; Hermans, P.W.M.; Mitchell, T.; Brown, J.S.

    2012-01-01

    Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the

  9. Expression of deleted in malignant brain tumor-1 (DMBT1) molecule in biliary epithelium is augmented in hepatolithiasis: possible participation in lithogenesis

    DEFF Research Database (Denmark)

    Sasaki, Motoko; Huang, Shiu-Feng; Chen, Miin-Fu

    2003-01-01

    Deleted in malignant brain tumor-1 (DMBT1) is a mucin-like molecule participating in mucosal immune defense. Given that bovine gallbladder mucin, which accelerates cholesterol crystallization, is a DMBT1 homolog, DMBT1 expression was examined immunohistochemically in biliary epithelial cells...

  10. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    International Nuclear Information System (INIS)

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B.

    1990-01-01<