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Sample records for cell rearrangements guided

  1. Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis.

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    Gabriel G Martins

    Full Text Available Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

  2. Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila.

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    Chihara, Takahiro; Kato, Kagayaki; Taniguchi, Misako; Ng, Julian; Hayashi, Shigeo

    2003-04-01

    Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. We used the tracheal system of Drosophila to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. We found that a reduced Rac activity led to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibited dynamic epithelial cell rearrangement. Hyperactivation of Rac, on the other hand, inhibited assembly of newly synthesized E-Cadherin into cell junctions and caused loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules.

  3. New type of cells with multiple chromosome rearrangements

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    Aseeva, Elena A. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation); Snigiryova, Galina P. [Russian Scientific Centre of Roentgenology and Radiology, ul. Profsoyuznaya 86, 117997 Moscow (Russian Federation); Neverova, Anna L. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation); Bogomazova, Alexandra N.; Novitskaya, Natalia N.; Khazins, Eva D. [Russian Scientific Centre of Roentgenology and Radiology, ul. Profsoyuznaya 86, 117997 Moscow (Russian Federation); Domracheva, Elena V. [National Research Centre for Hematology, Russian Academy of Medical Sciences, Novozykovsky proezd 4a, 125167 Moscow (Russian Federation)], E-mail: dom@blood.ru

    2010-04-15

    A comparative analysis of the distribution and the frequency of multiaberrant cells (MAC) among lymphocytes in different categories of low dose (up to 0.5 Gy) irradiated people was carried out. The highest MAC frequency was observed in people exposed to {alpha}-radiation (Pu, Rn) and in cosmonauts. This fact allows MAC to be considered as an indicator of a high-energy local exposure. A new type of cells with multiple chromosome rearrangements was discovered in the course of analysis of stable aberrations by the fluorescence in situ hybridization (FISH) method. The biological consequences of MAC formation and possibility of revealing the whole diversity of cells with multiple aberrations by means of modern molecular-cytogenetic methods are discussed.

  4. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

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    Kendra ePesko

    2015-04-01

    Full Text Available Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wildtype gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense nonsegmented RNA viruses (order Mononegavirales have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV variants with the nucleocapsid (N gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N-gene translocation towards the 5’ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function, especially on PC3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective

  5. Collective dynamics of cell migration and cell rearrangements

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    Kabla, Alexandre

    Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics. Geometrical confinement is one of the key external factors influencing large scale coordination during collective migration. Using a combination of in vitro experiments and numerical simulations, we show that the velocity correlation length, measured in unconfined conditions, provides a convenient length scale to predict the dynamic response under confinement. The same length scale can also be used to quantify the influence range of directional cues within the cell population. Heterogeneity within motile cell populations is frequently associated with an increase in their invasive capability and appears to play an important role during cancer metastasis. Using in silico experiments, we studied the way cell invasion is influenced by both the degree of cell coordination and the amount of variability in the motile force of the invading cells. Results suggest that mechanical heterogeneity dramatically enhances the invasion rate through an emerging cooperative process between the stronger and weaker cells, accounting for a number of observed invasion phenotypes. Effective convergent extension requires on a consistent orientation of cell intercalation at the tissue scale, most often in relation with planar cell polarity mechanisms to define the primary axes of deformation. Using a novel modelling approach for cells mechanical interactions, we studied the dynamics of substrate free motile cell populations. Ongoing work shows in particular that nematic order emerges

  6. Investigation of T-cell receptor-γ gene rearrangement in gastrointestinal lymphomas by PCR-SSCP analysis

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    Xi-Qun Han; Li He; Lan-Ying Shong; Hui-Yong Jiang; Mei-Gang Zhu; Tong Zhao

    2004-01-01

    AIM: To analyze the characterization of T-cell receptor-γ (TCR-γ) gene rearrangement in the gastrointestinal lymphomas and evaluate the value of PCR-SSCP analysis in gastrointestinal lymphomas investigation.METHODS: TCR-γgene rearrangement segments of gastrointestinal lymphomas were cloned and sequenced.Single clone plasmid and mixed clone plsamids were subsequently submitted to PCR-SSCP analysis to investigate the relationship between the number of amplified clones and band patterns of the amplified products. The PCR products of TCR-γgene rearrangement of 40 gastrointestinal lymphomas were electrophoresed on agarose gels and the positive cases on agarose gels were studied by SSCP analysis.RESULTS: The sequencing showed that TCR-γ gene rearrangement of the gastrointestinal lymphomas included functional gene and pseudogene with extensive variety in the junctional regions. In SSCP analysis, the number of the single-stranded bands was about two times of the number of amplified clones, and double-stranded band became broad with the increased number of the amplified clones. Thirteen of the 25 B-cell gastrointestinal lymphomas and 14 of the 15 gastrointestinal T-cell lymphomas were positive detected on agarose gel electrophoresis. Of the positive cases detected by SSCP analysis, 3 B-cell lymphomas and 13 T-cell lymphomas showed positive bands. The other cases showed only smears. The rearranged pattern included 13 monoallelic gene rearrangements and 3 biallelic or oligoclonal gene rearrangements.CONCLUSION: The pattern of TCR-γ, gene rearrangement in gastrointestinal lymphomas are similar to that of the nodular lymphomas. PCR-SSCP analysis for TCR-γ gene rearrangement can be applied both for adjuvant diagnosis of gastrointestinal lymphomas and analysis of the gene rearrangement pattern. The ratio of TCR-γ gene rearrangements occurred in T-cell gastrointestinal lymphomas is significantly higher than that in B-cell gastrointestinal lymphomas. The gene rearrangement

  7. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

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    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it po

  8. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

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    Mai Nanya

    Full Text Available Etoposide, a topoisomerase 2 (TOP2 inhibitor, is associated with the development of KMT2A (MLL-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

  9. Rapid, Nonradioactive Detection of Clonal T-Cell Receptor Gene Rearrangements in Lymphoid Neoplasms

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    Bourguin, Anne; Tung, Rosann; Galili, Naomi; Sklar, Jeffrey

    1990-11-01

    Southern blot hybridization analysis of clonal antigen receptor gene rearrangements has proved to be a valuable adjunct to conventional methods for diagnosing lymphoid neoplasia. However, Southern blot analysis suffers from a number of technical disadvantages, including the time necessary to obtain results, the use of radioactivity, and the susceptibility of the method to various artifacts. We have investigated an alternative approach for assessing the clonality of antigen receptor gene rearrangements in lymphoid tissue biopsy specimens. This approach involves the amplification of rearranged γ T-cell receptor genes by the polymerase chain reaction and analysis of the polymerase chain reaction products by denaturing gradient gel electrophoresis. By use of this approach, clonal rearrangements from neoplastic lymphocytes constituting as little as 0.1-1% of the total cells in the tissue are detected as discrete bands in the denaturing gel after the gel is stained with ethidium bromide and viewed under ultraviolet light. In contrast, polyclonal rearrangements from reactive lymphocytes appear as a diffuse smear along the length of the gel. Our findings suggest that polymerase chain reaction combined with denaturing gradient gel electrophoresis may offer a rapid, nonradioactive, and sensitive alternative to Southern blot analysis for the diagnostic evaluation of lymphoid tissue biopsy specimens.

  10. [Analyses of the rearrangement of T-cell receptor- and immunoglobulin genes in the diagnosis of lymphoproliferative disorders].

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    Griesser, D H

    1995-01-01

    Rearrangements are developmentally regulated genetic recombinations in T and B cells which generate functional T cell receptor (TcR) and immunoglobulin genes, respectively. Different variable, sometimes diversity, and joining gene segments which are discontinuously spread out within their chromosomal location in germline configuration, are randomly assembled in individual lymphocytes. These rearrangements can be detected by Southern Blot analysis if more than 5% of a total lymphocyte population in a biopsy specimen carries the same clonal rearrangement. We analyzed DNA from 324 snap-frozen biopsy specimens from lympho-proliferative disorders. None of the 20 reactive lesions and four malignant myelomonocytic tumors had a clonal antigen receptor gene rearrangement. All 117 malignant B cell lymphomas of different subtypes and 95 of 97 malignant T cell lymphomas showed a clonal gene rearrangement. Only two angioimmunoblastic lymphadenopathy(AILD)-type T cell lymphomas did not have immune receptor gene rearrangements. They were morphologically indistinguishable from the other 47 T/AILD lymphomas with clonal rearrangement patterns. In most cases TcR beta and immunoglobulin heavy chain (IgH) gene probes were sufficient for lineage assignment of the clonal T or B lymphocyte population. In 18% of B lymphomas, however, a cross-lineage rearrangement of TcR beta genes, and in 20% of the T cell lymphomas a clonal IgH gene rearrangement was detected. After exclusion of centrocytic, large cell anaplastic lymphomas (LCAL) of B-type, and T/AILD lymphomas which are overrepresented in our study, only 10% of the remaining 147 T and B cell lymphomas had aberrant rearrangements. TcR rearrangements other than those of the beta chain genes were extremely rare in B cell lymphomas, as were Ig kappa rearrangements in T lymphomas. Only two T/AILD lymphomas had IgH and Ig kappa rearrangement in addition to their clonal T cell receptor gene rearrangements. Both samples likely contain a clonal B

  11. Characterization of gene rearrangements resulted from genomic structural aberrations in human esophageal squamous cell carcinoma KYSE150 cells.

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    Hao, Jia-Jie; Gong, Ting; Zhang, Yu; Shi, Zhi-Zhou; Xu, Xin; Dong, Jin-Tang; Zhan, Qi-Min; Fu, Song-Bin; Wang, Ming-Rong

    2013-01-15

    Chromosomal rearrangements and involved genes have been reported to play important roles in the development and progression of human malignancies. But the gene rearrangements in esophageal squamous cell carcinoma (ESCC) remain to be identified. In the present study, array-based comparative genomic hybridization (array-CGH) was performed on the ESCC cell line KYSE150. Eight disrupted genes were detected according to the obviously distinct unbalanced breakpoints. The splitting of these genes was validated by dual-color fluorescence in-situ hybridization (FISH). By using rapid amplification of cDNA ends (RACE), genome walking and sequencing analysis, we further identified gene disruptions and rearrangements. A fusion transcript DTL-1q42.2 was derived from an intrachromosomal rearrangement of chromosome 1. Highly amplified segments of DTL and PTPRD were self-rearranged. The sequences on either side of the junctions possess micro-homology with each other. FISH results indicated that the split DTL and PTPRD were also involved in comprising parts of the derivative chromosomes resulted from t(1q;9p;12p) and t(9;1;9). Further, we found that regions harboring DTL (1q32.3) and PTPRD (9p23) were also splitting in ESCC tumors. The data supplement significant information on the existing genetic background of KYSE150, which may be used as a model for studying these gene rearrangements.

  12. Rearrangements of MYC gene facilitate risk stratification in diffuse large B-cell lymphoma patients treated with rituximab-CHOP

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    Tzankov, Alexandar; Xu-Monette, Zijun Y; Gerhard, Marc;

    2014-01-01

    In order to address the debatable prognostic role of MYC rearrangements in diffuse large B-cell lymphoma patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, we evaluated MYC rearrangements by fluorescence in situ hybridization in 563 cases using br...

  13. Clonal rearrangements of immunoglobulin genes and progression to B cell lymphoma in cutaneous lymphoid hyperplasia.

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    Wood, G S; Ngan, B Y; Tung, R; Hoffman, T E; Abel, E A; Hoppe, R T; Warnke, R A; Cleary, M L; Sklar, J

    1989-07-01

    Cutaneous lymphoid hyperplasia (CLH) is a disorder characterized by the development of one or more skin lesions containing dense lymphoid infiltrates that exhibit the histopathologic features of a benign, reactive process. Nevertheless, some cases have been associated with the subsequent development of clinically overt lymphomas. This suggests that monoclonal populations may exist in some cases of CLH and that these cases may represent a subset more likely to evolve into lymphoma. To determine if such a subset of CLH can be distinguished, Southern blot analysis of DNA was used to study the immunogenotypic features of lesions from 14 patients with clinical, histopathologic, and immunopathologic findings characteristic of CLH. Five cases exhibited detectable clonal rearrangements of immunoglobulin genes. Furthermore, one of these five cases evolved into overt diffuse large cell lymphoma of B cell lineage during a 2-year follow-up of recurrent disease at the original cutaneous site. The immunoglobulin gene rearrangements of this lymphoma were identical to those of the prior CLH lesion. There was no evidence of detectable t(14;18) chromosomal translocations or clonal rearrangements of the beta gene of the T cell receptor in any case. It was concluded that CLH can be divided into two subsets based on the presence or absence of a clonal B cell population, and that overt lymphoma can arise from the former subset and contain the same B cell clone identified in the pre-existent CLH lesion.

  14. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

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    Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

    2015-03-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

  15. Deletional rearrangement in the human T-cell receptor. cap alpha. -chain locus

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

    1987-12-01

    The antigen-specific receptor on the surface of mature T lymphocytes is a heterodimer consisting of polypeptides termed ..cap alpha.. and ..beta... In the course of characterizing human T-cell tumors with an immature (CD4/sup -/, CD8/sup -/) surface phenotype, the authors detected a 2-kilobase ..cap alpha..-related transcript. Analysis of cDNA clones corresponding to this transcript established that a genetic element (which they call TEA, for T early ..cap alpha..) located between the ..cap alpha..-chain variable- and joining-region genes had been spliced to the ..cap alpha.. 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 ..cap alpha.. 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 ..cap alpha.. gene with its complex diversity. These DNA blots also showed that TEA is generally present in the germ-line configuration in cells expressing the ..gamma..delta heterodimeric receptor and is deleted from mature (..cap alpha beta..-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 (..gamma..delta-expressing) T cells and mature (..cap alpha beta..-expressing) T cells.

  16. Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

    OpenAIRE

    2007-01-01

    The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the ...

  17. Cytoskeleton rearrangement induced by tetraspanin engagement modulates the activation of T and NK cells.

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    Crotta, Stefania; Ronconi, Vanessa; Ulivieri, Cristina; Baldari, Cosima T; Valiante, Nicholas M; Valiente, Nicholas M; Abrignani, Sergio; Wack, Andreas

    2006-04-01

    The hepatitis C virus (HCV) binds to human cells through the interaction of its envelope glycoprotein E2 with the tetraspanin CD81. We have previously reported that engagement of CD81 has opposite effects on T and NK cell function, as it enhances T cell receptor-mediated T cell activation and inhibits CD16- or IL-12-mediated NK cell activation. We further investigated this dichotomy and found that another tetraspanin, CD82, induces the same opposing effects on human primary T and NK cells. Activation by other unrelated stimuli such as NKG2D- and beta-1 integrin is also reduced by CD81 ligation on NK cells. CD81 engagement by monoclonal antibody or HCV-E2 enhances zeta and Erk phosphorylation in T cells and reduces them in NK cells, reflecting the opposite functional outcomes. CD81 engagement induces dramatic morphological changes and local F-actin accumulation in both NK and T cells, indicating rearrangement of the actin cytoskeleton. Pharmacological inhibition of actin polymerization reduces T cell activation, whereas it greatly enhances NK cell activation. Importantly, treatment with actin blockers abolishes the inhibitory effect of CD81 ligation on NK cells. We propose that tetraspanin engagement leads to comparable cytoskeleton reorganization in T and NK cells, which in turn results in opposite functional outcomes.

  18. Extensive junctional diversity of rearranged human T cell receptor delta genes.

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    Hata, S; Satyanarayana, K; Devlin, P; Band, H; McLean, J; Strominger, J L; Brenner, M B; Krangel, M S

    1988-06-10

    The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.

  19. Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS.

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    Ma, Long; Yang, Liwen; Bin Shi; He, Xiaoyan; Peng, Aihua; Li, Yuehong; Zhang, Teng; Sun, Suhong; Ma, Rui; Yao, Xinsheng

    2016-07-12

    V-D-J rearrangement of the TCR-beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR-beta chain V-J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR-beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differed from that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.

  20. REARRANGEMENT AND EXPRESSION OF T CELL RECEPTOR β GENE IN HUMAN HEMOPOIETIC CELL LINES AND PRIMARY CELLS FROM ACUTE LYMPHOCYTIC LEUKEMIAS

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    仇一华; 陈诗书

    1992-01-01

    Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia (ALL) patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with 32P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.

  1. Rearrangements of chicken immunoglobulin genes in lymphoid cells transformed by the avian retroviral oncogene v-rel.

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    Chen, L; Lim, M Y; Bose, H; Bishop, J M

    1988-01-01

    The retroviral oncogene v-rel transforms poorly characterized lymphoid cells. We have explored the nature of these cells by analyzing the configuration and expression of immunoglobulin genes in chicken hemopoietic cells transformed by v-rel. None of the transformed cells expressed their immunoglobulin genes. The cells fell into three classes: class I cells have their immunoglobulin genes potentially in an embryonic configuration; class II and class III cells have lost one copy of the lambda light chain locus and have one copy of the heavy chain locus rearranged into a configuration that differs from what is found in mature B cells. In class II cells, the other heavy chain locus may be in embryonic configuration, whereas it is deleted in class III cells. The first of these classes may represent the earliest stage of the lymphoid lineage yet encountered among virus-transformed cells, whereas the second and third classes represent an apparently anomalous rearrangement whose origin remains unknown.

  2. Rearrangements between differentiating hair cells coordinate planar polarity and the establishment of mirror symmetry in lateral-line neuromasts

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    Ivana Mirkovic

    2012-04-01

    In addition to their ubiquitous apical-basal polarity, many epithelia are also polarized along an orthogonal axis, a phenomenon termed planar cell polarity (PCP. In the mammalian inner ear and the zebrafish lateral line, PCP is revealed through the orientation of mechanosensitive hair cells relative to each other and to the body axes. In each neuromast, the receptor organ of the lateral line, hair bundles are arranged in a mirror-symmetrical fashion. Here we show that the establishment of mirror symmetry is preceded by rotational rearrangements between hair-cell pairs, a behavior consistently associated with the division of hair-cell precursors. Time-lapse imaging of trilobite mutants, which lack the core PCP constituent Vang-like protein 2 (Vangl2, shows that their misoriented hair cells correlate with misaligned divisions of hair-cell precursors and an inability to complete rearrangements accurately. Vangl2 is asymmetrically localized in the cells of the neuromast, a configuration required for accurate completion of rearrangements. Manipulation of Vangl2 expression or of Notch signaling results in a uniform hair-cell polarity, indicating that rearrangements refine neuromast polarity with respect to the body axes.

  3. A subset of prostatic basal cell carcinomas harbor the MYB rearrangement of adenoid cystic carcinoma.

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    Bishop, Justin A; Yonescu, Raluca; Epstein, Jonathan I; Westra, William H

    2015-08-01

    Adenoid cystic carcinoma (ACC) is a basaloid tumor consisting of myoepithelial and ductal cells typically arranged in a cribriform pattern. Adenoid cystic carcinoma is generally regarded as a form of salivary gland carcinoma, but it can arise from sites unassociated with salivary tissue. A rare form of prostate carcinoma exhibits ACC-like features; it is no longer regarded as a true ACC but rather as prostatic basal cell carcinoma (PBCC) and within the spectrum of basaloid prostatic proliferations. True ACCs often harbor MYB translocations resulting in the MYB-NFIB fusion protein. MYB analysis could clarify the true nature of prostatic carcinomas that exhibit ACC features and thus help refine the classification of prostatic basaloid proliferations. Twelve PBCCs were identified from the pathology consultation files of Johns Hopkins Hospital. The histopathologic features were reviewed, and break-apart fluorescence in situ hybridization for MYB was performed. All 12 cases exhibited prominent basaloid histology. Four were purely solid, 7 exhibited a cribriform pattern reminiscent of salivary ACC, and 1 had a mixed pattern. The MYB rearrangement was detected in 2 (29%) of 7 ACC-like carcinomas but in none (0%) of the 5 PBCCs with a prominent solid pattern. True ACCs can arise in the prostate as is evidenced by the presence of the characteristic MYB rearrangement. When dealing with malignant basaloid proliferations in the prostate, recommendations to consolidate ACCs with other tumor types may need to be reassessed, particularly in light of the rapidly advancing field of biologic therapy where the identification of tumor-specific genetic alterations presents novel therapeutic targets.

  4. Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

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    Alvaro Muñoz-López

    2016-10-01

    Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

  5. T-cell receptor gene rearrangement analysis: cutaneous T cell lymphoma, peripheral T cell lymphoma, and premalignant and benign cutaneous lymphoproliferative disorders.

    Science.gov (United States)

    Zelickson, B D; Peters, M S; Muller, S A; Thibodeau, S N; Lust, J A; Quam, L M; Pittelkow, M R

    1991-11-01

    T-cell receptor gene rearrangement analysis is a useful technique to detect clonality and determine lineage of lymphoid neoplasms. We examined 103 patients with mycosis fungoides, Sézary syndrome, peripheral T cell lymphoma, potentially malignant lymphoproliferative disorders including pre-Sézary syndrome, large plaque parapsoriasis, lymphomatoid papulosis and follicular mucinosis, and various benign inflammatory infiltrates. A clonal rearrangement was detected in skin samples in 20 of 24 patients with mycosis fungoides and in peripheral blood samples in 19 of 21 patients with Sézary syndrome. A clonal population was also detected in seven of eight cases classified as peripheral T cell lymphoma. The potentially malignant dermatoses tended to have clonal rearrangement, with the exception of large plaque parapsoriasis, and further follow-up is needed to correlate clonality with the disease course. These studies demonstrate the value of molecular genetics as an adjunct to morphology in the examination of patients with cutaneous lymphoproliferative disease.

  6. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  7. Diagnosis of mantle cell lymphoma and detection of bcl-1 gene rearrangement

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Sook; Cho, Kyung Ja; Lee, Sun Joo [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    We reclassified a large series of non-Hogkin`s lymphoma diagnosed at Korea Cancer Center Hospital from 1991 to 1995, according to REAL classification, and compared the efficacy of immunohistochemical study for cyclin D1 protein and PCR for bcl-1 gene rearrangement to diagnose mantle cell lymphoma (MCL). By REAL classification, 7 %, diffuse large B-cell lymphoma was the most common type (51.8%) and was followed by peripheral T-cell lymphoma-unspecified (10%) and angiocentric lymphoma (7.5%). The most reliable histologic finding was mitosis to make a differential diagnosis. Mitoses of MCL were 17/10 HPF in average and all the cases showed more than 10/10 HPF. Immunophenotypic study alone cannot lead to a differential diagnosis between MCL and SLL, and the overexpression of cyclin D1 was the most important for diagnosis of MCL . Both immunohistochemistry for cyclin D1 and PCR for bcl-1 were specific for MCL and immunohistochemistry was more sensitive than PCR. Statistical analysis showed a different survival rate between MCL and the other low-grade B-cell lymphomas (SLL + MALT + LPL) and a difference between MCL and SLL. Immunohistochemical detection of cyclin D1 has a practical usefulness in making routine diagnosis of MCL. The initial accurate diagnosis of MCL will help clinicians make a proper management. (author). 27 refs., 6 tabs., 4 figs.

  8. Inhibition of RORγT Skews TCRα Gene Rearrangement and Limits T Cell Repertoire Diversity

    Directory of Open Access Journals (Sweden)

    Yanxia Guo

    2016-12-01

    Full Text Available Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and T cell receptor (TCRα selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits T cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on T cell biology.

  9. Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

    NARCIS (Netherlands)

    Droese, J.; Langerak, A.W.; Groenen, P.J.T.A.; Bruggemann, M.; Neumann, P.; Wolvers-Tettero, I.L.M.; Altena, M.C. van; Kneba, M.; Dongen, J.J.M. van

    2004-01-01

    The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB)

  10. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma

    NARCIS (Netherlands)

    Kramer, M.H.H.; Hermans, J; Wijburg, E; Philippo, K; Geelen, E; van Krieken, J.H.J.M.; de Jong, D; Maartense, E; Schuuring, E; Kluin, P M

    1998-01-01

    Diffuse large B-cell lymphoma (DLCL) is characterized by a marked degree of morphologic and clinical heterogeneity. We studied 156 patients with de novo DLCL for rearrangements of the BCL2, BCL6, and MYC oncogenes by Southern blot analysis and BCL2 protein expression. We related these data to the pr

  11. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xionggao [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Department of Ophthalmology, Hainan Medical College, Haikou (China); Wei, Yantao; Ma, Haizhi [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Zhang, Shaochong, E-mail: zhshaochong@163.com [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  12. Construction of BAC-based physical map and analysis of chromosome rearrangement in Chinese hamster ovary cell lines.

    Science.gov (United States)

    Cao, Yihua; Kimura, Shuichi; Itoi, Takayuki; Honda, Kohsuke; Ohtake, Hisao; Omasa, Takeshi

    2012-06-01

    Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines.

  13. Discrete Element Modeling Results of Proppant Rearrangement in the Cooke Conductivity Cell

    Energy Technology Data Exchange (ETDEWEB)

    Earl Mattson; Hai Huang; Michael Conway; Lisa O' Connell

    2014-02-01

    The study of propped fracture conductivity began in earnest with the development of the Cooke cell which later became part of the initial API standard. Subsequent developments included a patented multicell design to conduct 4 tests in a press at the same time. Other modifications have been used by various investigators. Recent studies by the Stim-Lab proppant consortium have indicated that the flow field across a Cooke proppant conductivity testing cell may not be uniform as initially believed which resulted is significantly different conductivity results. Post test analysis of low temperature metal alloy injections at the termination of proppant testing prior to the release of the applied stress suggest that higher flow is to be expected along the sides and top of the proppant pack than compared to the middle of the pack. To evaluate these experimental findings, a physics-based two-dimensional (2-D) discrete element model (DEM) was developed and applied to simulate proppant rearrangement during stress loading in the Cooke conductivity cell and the resulting porosity field. Analysis of these simulations are critical to understanding the impact of modification to the testing cell as well as understanding key proppant conductivity issues such as how these effects are manifested in proppant concentration testing results. The 2-D DEM model was constructed to represent a realistic cross section of the Cooke cell with a distribution of four material properties, three that represented the Cooke cell (steel, sandstone,square rings), and one representing the proppant. In principle, Cooke cell materials can be approximated as assemblies of independent discrete elements (particles) of various sizes and material properties that interact via cohesive interactions, repulsive forces, and frictional forces. The macroscopic behavior can then be modeled as the collective behavior of many interacting discrete elements. This DEM model is particularly suitable for modeling proppant

  14. Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells.

    Science.gov (United States)

    Averbeck, Marco; Braun, Thorsten; Pfeifer, Gunther; Sleeman, Jonathan; Dudda, Jan; Martin, Stefan F; Kremer, Bernhard; Aktories, Klaus; Simon, Jan C; Termeer, Christian

    2004-10-01

    The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.

  15. Rearrangement and expression of beta-T-cell receptor and immunoglobulin genes in established Ph1 chronic myelogenous leukemia cell lines.

    Science.gov (United States)

    Berenson, J; Koeffler, H P

    1989-01-01

    We have determined the arrangement and expression of immunoglobulin (Ig) and beta-T-cell receptor (TCR) genes in six established Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML) cell lines, and correlated these results with their phenotypic characteristics. Three cell lines with nonlymphoid characteristics, EM2, EM3, and K562, did not demonstrate rearrangement or expression of Ig or beta-TCR genes. A new cell line, MB, with a mature B-cell phenotype recently established in our laboratory, contained light and heavy chain immunoglobulin gene rearrangements and expressed mature Ig RNA. In a cell line with an early lymphoid phenotype, BV173, this analysis showed rearrangement of Ig heavy chain and beta-TCR genes, unrearranged Ig light chain DNA, and expression of only an immature beta-TCR transcript. This line provides evidence for T-cell lineage involvement in Ph1 CML. One cell line without markers of any cell type, KCL-22, demonstrated rearranged, unexpressed Ig heavy chain genes, suggesting these cells are at the very earliest stages of lymphoid differentiation. These lines should provide valuable tools to dissect the molecular biology of differentiation in CML and in early lymphocytes.

  16. CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability

    Science.gov (United States)

    Inoue, Takeshi; Morita, Masahiro; Hijikata, Atsushi; Fukuda-Yuzawa, Yoko; Adachi, Shungo; Isono, Kyoichi; Ikawa, Tomokatsu; Kawamoto, Hiroshi; Koseki, Haruhiko; Natsume, Tohru; Fukao, Taro; Ohara, Osamu; Yamamoto, Tadashi

    2015-01-01

    The CCR4–NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre–B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4–NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA. PMID:26238124

  17. Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

    Science.gov (United States)

    Whetsell, M; Mosley, R L; Whetsell, L; Schaefer, F V; Miller, K S; Klein, J R

    1991-12-01

    The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

  18. Consistency of VDJ Rearrangement and Substitution Parameters Enables Accurate B Cell Receptor Sequence Annotation.

    Directory of Open Access Journals (Sweden)

    Duncan K Ralph

    2016-01-01

    Full Text Available VDJ rearrangement and somatic hypermutation work together to produce antibody-coding B cell receptor (BCR sequences for a remarkable diversity of antigens. It is now possible to sequence these BCRs in high throughput; analysis of these sequences is bringing new insight into how antibodies develop, in particular for broadly-neutralizing antibodies against HIV and influenza. A fundamental step in such sequence analysis is to annotate each base as coming from a specific one of the V, D, or J genes, or from an N-addition (a.k.a. non-templated insertion. Previous work has used simple parametric distributions to model transitions from state to state in a hidden Markov model (HMM of VDJ recombination, and assumed that mutations occur via the same process across sites. However, codon frame and other effects have been observed to violate these parametric assumptions for such coding sequences, suggesting that a non-parametric approach to modeling the recombination process could be useful. In our paper, we find that indeed large modern data sets suggest a model using parameter-rich per-allele categorical distributions for HMM transition probabilities and per-allele-per-position mutation probabilities, and that using such a model for inference leads to significantly improved results. We present an accurate and efficient BCR sequence annotation software package using a novel HMM "factorization" strategy. This package, called partis (https://github.com/psathyrella/partis/, is built on a new general-purpose HMM compiler that can perform efficient inference given a simple text description of an HMM.

  19. Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement.

    Directory of Open Access Journals (Sweden)

    Mikihito Kajiya

    Full Text Available The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29, a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+/OmpA(- E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9 than Omp29(-/OmpA(- E. coli. While the entry of Aa and Omp29(+/OmpA(- E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK, a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin and Rho GTPases inhibitor (EDIN, both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

  20. Laser-based microdissection of single cells from tissue sections and PCR analysis of rearranged immunoglobulin genes from isolated normal and malignant human B cells.

    Science.gov (United States)

    Küppers, Ralf; Schneider, Markus; Hansmann, Martin-Leo

    2013-01-01

    Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene family-specific primers recognizing nearly all V gene segments together with primers for the J gene segments. By sequence analysis of V genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

  1. Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells

    NARCIS (Netherlands)

    Pohjoismaki, J.L.; Goffart, S.; Spelbrink, J.N.

    2011-01-01

    Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively d

  2. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

    Science.gov (United States)

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

    2006-04-21

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

  3. Clear cell myoepithelial carcinoma of salivary glands showing EWSR1 rearrangement: molecular analysis of 94 salivary gland carcinomas with prominent clear cell component.

    Science.gov (United States)

    Skálová, Alena; Weinreb, Ilan; Hyrcza, Martin; Simpson, Roderick H W; Laco, Jan; Agaimy, Abbas; Vazmitel, Marina; Majewska, Hanna; Vanecek, Tomas; Talarčik, Peter; Manajlovic, Spomenka; Losito, Simona N; Šteiner, Petr; Klimkova, Adela; Michal, Michal

    2015-03-01

    This study examines the presence of the EWSR1 rearrangement in a variety of clear cell salivary gland carcinomas with myoepithelial differentiation. A total of 94 salivary gland carcinomas with a prominent clear cell component included 51 cases of clear cell myoepithelial carcinomas de novo (CCMC), 21 cases of CCMCs ex pleomorphic adenoma (CCMCexPA), 11 cases of epithelial-myoepithelial carcinoma (EMC), 6 cases of EMC with solid clear cell overgrowth, and 5 cases of hyalinizing clear cell carcinoma of minor salivary glands. In addition, 10 cases of myoepithelial carcinomas devoid of clear cell change and 12 cases of benign myoepithelioma were included as well. All the tumors in this spectrum were reviewed, reclassified, and tested by fluorescence in situ hybridization (FISH) for the EWSR1 rearrangement using the Probe Vysis EWSR1 Break Apart FISH Probe Kit. The EWSR1 rearrangement was detected in 20 of 51 (39%) cases of CCMC, in 5 of 21 (24%) cases of CCMCexPA, in 1 of 11 (9%) cases of EMC, and in 4 of 5 (80%) cases of hyalinizing clear cell carcinoma. The 25 EWSR1-rearranged CCMCs and CCMCexPAs shared similar histomorphology. They were arranged in nodules composed of compact nests of large polyhedral cells with abundant clear cytoplasm. Necrosis, areas of squamous metaplasia, and hyalinization were frequent features. Immunohistochemically, the tumors expressed p63 (96%), cytokeratin CK14 (96%), and S100 protein (88%). MIB1 index varied from 10% to 100%, with most cases in the 20% to 40% range. Clinical follow-up information was available in 21 cases (84%) and ranged from 3 months to 15 years (mean 5.2 y); 4 patients were lost to follow-up. Ten patients are alive with no evidence of recurrent or metastatic disease in the follow-up period from 3 months to 15 years (mean 5 y), 3 patients are alive with recurrent and metastatic disease, and 8 died of disseminated cancer 9 months to 16 years after diagnosis (mean 6 y). Lymph node metastasis appeared in 5 patients

  4. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.

    Science.gov (United States)

    Berdoz, J; Monath, T P; Kraehenbuhl, J P

    1995-04-01

    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  5. Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies

    Science.gov (United States)

    Mouka, Aurélie; Izard, Vincent; Tachdjian, Gérard; Brisset, Sophie; Yates, Frank; Mayeur, Anne; Drévillon, Loïc; Jarray, Rafika; Leboulch, Philippe; Maouche-Chrétien, Leila; Tosca, Lucie

    2017-01-01

    Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development. PMID:28045072

  6. Clinicopathologic characteristics andtherapeutic responses ofChinese patients withnon-small cell lung cancer who harbor an anaplastic lymphoma kinase rearrangement

    Institute of Scientific and Technical Information of China (English)

    ShaFu; HaiYunWang; FangWang; MaYanHuang; LingDeng; XiaoZhang; ZuLuYe; JianYong Shao

    2015-01-01

    Introduction:The rearrangement of the anaplastic lymphoma kinase (ALK) gene accounts for approximately 1%–6%of lung adenocarcinoma cases and deifnes a molecular subgroup of tumors characterized by clinical sensitivity toALK inhibitors such as crizotinib. This study aimed to identify the relationship betweenALK rearrangement and the clinico‑pathologic characteristics of non‑small cell lung cancer (NSCLC) and to analyze the therapeutic responses of crizotinib and conventional chemotherapy toALK rearrangement in NSCLC patients. Methods:A total of 487 lung cancer patients who underwent testing forALK rearrangement in our department were included in this study.ALK rearrangement was examined by using lfuorescence insitu hybridization (FISH) assay. Results:Among the 487 patients, 44 (9.0%) were diagnosed withALK rearrangement by using FISH assay. In 123 patients with adenocarcinoma who were non‑smokers and of a young age (≤58years old), the frequency ofALK rearrangement was 20.3% (25/123). Short overall survival (OS) was associated with non‑adenocarcinoma tumor type (P=0.006), poorly differentiated tumors (P=0.001), advanced‑stage tumors (P<0.001), smoking history (P=0.008), and wild‑type epidermal growth factor receptor (EGFR) (P=0.008). Moreover, patients with poorly differentiated and advanced‑stage tumors had a shorter time to cancer progression compared with those with well differentiated (P=0.023) and early‑stage tumors (P=0.001), respectively. Conclusions:ALK‑rearranged NSCLC tends to occur in younger individuals who are either non‑smokers or light smokers with adenocarcinoma. Patients withALK rearrangement might beneift fromALK inhibitor therapy.

  7. Coordinated rearrangements of assimilatory and storage cell compartments in a nitrogen-starving symbiotic chlorophyte cultivated under high light.

    Science.gov (United States)

    Gorelova, Olga; Baulina, Olga; Solovchenko, Alexei; Selyakh, Irina; Chivkunova, Olga; Semenova, Larisa; Scherbakov, Pavel; Burakova, Olga; Lobakova, Elena

    2015-03-01

    A quantitative micromorphometric study of the cell compartment rearrangements was performed in a symbiotic chlorophyte Desmodesmus sp. 3Dp86E-1 grown on nitrogen (N) replete or N-free medium under 480 μmol PAR quanta m(-2) s(-1). The changes in the chloroplast, intraplastidial, and cytoplasmic inclusions induced by high light (HL) and N starvation were similar to those characteristic of free-living chlorophytes. The N-sufficient culture responded to HL by a transient swelling of the thylakoid lumen and a decline in photosynthetic efficiency followed by its recovery. In the N-starving cells, a more rapid expansion and thylakoid swelling occurred along with the irreversible decline in the photosynthetic efficiency. Differential induction of starch grains, oil bodies, and cell wall polysaccharides depending on the stress exposure and type was recorded. Tight relationships between the changes in the assimilatory and storage compartments in the stressed Desmodesmus sp. cells were revealed.

  8. Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production.

    Science.gov (United States)

    Vela, José Luis; Aït-Azzouzene, Djemel; Duong, Bao Hoa; Ota, Takayuki; Nemazee, David

    2008-02-01

    The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

  9. Direct assessment of junctional diversity in rearranged T cell receptor β chain encoding genes by combined heteroduplex and single strand conformation polymorphism (SSCP) analysis

    NARCIS (Netherlands)

    Offermans, M.T.C.; Struyk, L.; Geus, B. de; Breedveld, F.C.; Elsen, P.J. van den; Rozing, J.

    1996-01-01

    In order to define the extent of T cell heterogeneity and clonality, unique DNA sequences in the junctional region in rearranged T cell receptor (TcR) genes can be studied. For this purpose we have adapted a non-denaturing nucleic acid gel electrophoresis procedure to detect TcR junctional diversity

  10. Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

    DEFF Research Database (Denmark)

    Christensen, M; Funder, A D; Bendix, K;

    2006-01-01

    AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods. METHODS: The DNA for PCR......% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient. CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however...

  11. Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement

    DEFF Research Database (Denmark)

    Zhou, X.G.; Sandvej, K.; Gregersen, Niels

    1999-01-01

    rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly...... because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present...

  12. A Systemic Review of Resistance Mechanisms and Ongoing Clinical Trials in ALK-rearranged Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Khashayar eEsfahani

    2014-07-01

    Full Text Available The identification of oncogenic driver driver mutations in non-small cell lung cancer has led to a paradigm shift and the development of specific molecular treatments. Tumors harboring a rearranged EML4-ALK fusion oncogene are highly sensitive to therapy with ALK-targeted inhibitors. Crizotinib is the first approved treatment for advanced lung tumors containing this genetic abnormality. In this mini review, we discuss the existing data on crizotinib as well as ongoing trials involving this medication. A brief overview of the known resistance mechanisms to criztotinib will also be presented followed by a summary of the ongoing trials involving next-generation ALK inhibitors or other targeted therapies in patients with ALK+ NSCLC.

  13. Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Shivang Doshi

    2016-01-01

    Full Text Available Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH and immunohistochemistry (IHC. However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies—IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe. The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies.

  14. Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA.

    Science.gov (United States)

    Oberle, Anna; Brandt, Anna; Voigtlaender, Minna; Thiele, Benjamin; Radloff, Janina; Schulenkorf, Anita; Alawi, Malik; Akyüz, Nuray; März, Manuela; Ford, Christopher T; Krohn-Grimberghe, Artus; Binder, Mascha

    2017-02-09

    Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for disease monitoring in patients with solid and hematological malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring of circulating myeloma cells (cmc-V(D)J) and cell-free myeloma DNA (cfm-V(D)J). Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for cmc-/cfm-V(D)J was associated with conventional remission status (pJ (pJ despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and therefore decline more rapidly after initiation of effective treatment. Positivity for cmc- and cfm-V(D)J was associated with each other (p=0.042), but in 30% discordant. This indicated that cfm-V(D)J may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.

  15. Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia

    NARCIS (Netherlands)

    Derksen, P W; Langerak, A W; Kerkhof, E; Wolvers-Tettero, I L; Boor, P P; Mulder, A H; Vrints, L W; Coebergh, J W; van Krieken, J H; Schuuring, E; Kluin, P M; van Dongen, J J

    1999-01-01

    Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality ass

  16. Alectinib for choroidal metastasis in a patient with crizotinib-resistant ALK rearranged positive non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Okuma Y

    2015-06-01

    Full Text Available Yusuke Okuma,1,2 Yuichiro Tanaka,3 Tina Kamei,1 Yukio Hosomi,1 Tatsuru Okamura1 1Department of Thoracic Oncology and Respiratory Medicine, Tokyo Metropolitan Cancer and Infectious diseases Center Komagome Hospital, 2Division of Oncology, Research Center for Medical Sciences, The Jikei University School of Medicine, 3Department of Ophthalmology, Tokyo Metropolitan Cancer and Infectious diseases Center Komagome Hospital, Tokyo, Japan Abstract: Choroidal metastasis is rare in cancer patients. Small molecules of molecular targeted agents for lung cancer with actionable mutations were reported to be palliated for symptoms caused by choroidal metastasis. Visual disturbance by choroidal metastasis significantly decreases quality of life during the patient’s remaining lifespan; therefore, radiotherapy or laser photocoagulation is proposed with consensus. However, improvement in survival with matched molecular targeted agents for oncogenic driver mutations reminds us to also be concerned with late treatment toxicities. A 30-year-old female patient previously treated with crizotinib harboring ALK rearranged non-small cell lung cancer complained of visual disturbance, fever, and bone pains undergoing anti-PD-1 antibody treatment. A decreased proportion of ALK fusion was demonstrated by fluorescence in situ hybridization in liver metastasis compared to the primary site in a chemo-naïve state. She was diagnosed with low vision, choroidal metastasis and retinal detachment. Therefore, she started alectinib treatment and both her ocular and systemic symptoms were palliated in a week. Later, she temporarily discontinued alectinib because of skin rash although the choroidal metastasis and retinal detachment resolved and she regained low vision completely at 2 weeks. She obtained partial response with alectinib for more than 5 months after recovering from skin rash. Keywords: lung cancer, ALK rearrangement, alectinib, choroidal metastasis, molecular targeted

  17. The utility of the in situ detection of T-cell receptor Beta rearrangements in cutaneous T-cell-dominant infiltrates.

    Science.gov (United States)

    Magro, Cynthia M; Nuovo, Gerard J; Crowson, A Neil

    2003-09-01

    The diagnostic assessment of cutaneous T-cell infiltrates is problematic for dermatopathologists. A variety of conditions, including lymphomatoid hypersensitivity reactions and lymphomatoid lupus erythematosus, can demonstrate lymphoid atypia and phenotypic changes that can mimic cutaneous T-cell lymphoma (CTCL). A similar issue revolves around lymphoid dyscrasias, which includes parapsoriasis, atypical pigmentary purpura, pityriasis lichenoides chronica, indeterminate lymphocytic lobular panniculitis, and lymphomatoid papulosis, which can progress to CTCL. A reverse transcription (RT) in situ PCR assay for T-cell receptor beta rearrangements (TCRbeta) was used to assess T-cell clonality in formalin-fixed, paraffin-embedded tissues. In 7 of 8 cases of classic CTCL, the RT in situ PCR assay for TCRbeta rearrangement showed monoclonality; the other was biclonal. Further, in cases with multiple lesions over time, the same T-cell clone could be detected including in those patients whose biopsies showed large-cell transformation. Monoclonality was also demonstrated in each of 2 cases of cutaneous lymphomatoid papulosis. Demonstration of oligoclonality (and one case of biclonality) by RT in situ PCR was confined to those cases that either represented prelymphomatous conditions such as large plaque parapsoriasis or pityriasis lichenoides or lesions of drug-induced lymphomatoid hypersensitivity that all demonstrated clinical regression. In conclusion, RT in situ PCR for TCRbeta, which can be done on formalin-fixed biopsies and allows direct correlation of the molecular data with the histology, is a useful adjunctive test in the differentiation of CTCL from its mimics.

  18. Clinicopathology, immunophenotype, T cell receptor gene rearrangement, Epstein-Barr virus status and p53 gene mutation of cutaneous extranodal NK/T-cell lymphoma, nasal-type

    Institute of Scientific and Technical Information of China (English)

    WANG Ting-ting; XU Chen; LIU Shan-ling; KAN Bei; RAN Yu-ping; LIU Wei-ping; LI Gan-di

    2013-01-01

    Background Extranodal natural killer/T-cell (NK/T cell) lymphoma,nasal-type,is a rare lymphoma.Skin is the second most common site of involvement after the nasal cavity/nasalpharynx.The aim of this study was to investigate the clinicopathologic features,immunophenotype,T cell receptor (TCR) gene rearrangement,the association with Epstein-Barr virus (EBV) infection and p53 gene mutations of the lymphoma.Methods The clinicopathologic analysis,immunohistochemistry,in situ hybridization for EBER1/2,TCR gene rearrangement by polymerase chain reaction (PCR),mutations of p53 gene analyzed by PCR and sequence analysis were employed in this study.Results In the 19 cases,the tumor primarily involved the dermis and subcutaneous layer.Immunohistochemical staining showed that most of the cases expressed CD45RO,CD56,CD3ε,TIA-1 and GrB.Three cases were positive for CD3 and two cases were positive for CD30.Monoclonal TCRY gene rearrangement was found in 7 of 18 cases.The positive rate of EBER1/2 was 100%.No p53 gene mutation was detected on the exon 4-9 in the 18 cases.Fifteen cases showed Pro (proline)/Arg (arginine) single nucleotide polymorphisms (SNPs) on the exon 4 at codon 72.The expression of p53 protein was 72% (13/18) immunohistochemically.Conclusions Cutaneous NK/T-cell lymphoma is a rare but highly aggressive lymphoma with poor prognosis.No p53 gene mutation was detected on the exon 4-9,and Pro/Arg SNPs on p53 codon 72 were detected in the cutaneous NK/T-cell lymphoma.The overexpression of p53 protein may not be the result of p53 gene mutation.

  19. Regulation of immunoglobulin gene rearrangement and expression.

    Science.gov (United States)

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching.

  20. Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

    Science.gov (United States)

    Cao, Zhiyi; Gyawali, Smita; Gong, Haiyan; Soza, Andrea; González, Alfonso; Panjwani, Noorjahan

    2012-01-01

    Purpose The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling. Methods Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β1 integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined. Principal Findings We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β1 integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632. Conclusions/Significance The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow. PMID:22973445

  1. Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

    NARCIS (Netherlands)

    Á. Muñoz-López (Álvaro); D. Romero-Moya (Damià); C. Prieto (Cristina); Ramos-Mejía, V. (Verónica); Agraz-Doblas, A. (Antonio); I. Varela (Ignacio); Buschbeck, M. (Marcus); Palau, A. (Anna); Carvajal-Vergara, X. (Xonia); Giorgetti, A. (Alessandra); Ford, A. (Anthony); M. Lako (Majlinda); Granada, I. (Isabel); Ruiz-Xivillé, N. (Neus); Rodríguez-Perales, S. (Sandra); Torres-Ruíz, R. (Raul); R.W. Stam (Ronald); Fuster, J.L. (Jose Luis); M.F. Fraga (Mario F.); Nakanishi, M. (Mahito); G. Cazzaniga (Gianni); Bardini, M. (Michela); Cobo, I. (Isabel); Bayon, G.F. (Gustavo F.); A.F. Fernández (Agustin F.); C. Bueno (C.); P. Menéndez (Pablo)

    2016-01-01

    textabstractInduced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies gen

  2. Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

    Energy Technology Data Exchange (ETDEWEB)

    Puxeddu, Efisio [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Zhao Guisheng [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Stringer, James R. [Department of Molecular Genetics, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Medvedovic, Mario [Center for Biostatistic Service, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Moretti, Sonia [Dipartimento di Medicina Interna, Universita degli Studi di Perugia, Via E. dal Pozzo, Perugia 06126, (Italy); Fagin, James A. [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States)]. E-mail: james.fagin@uc.edu

    2005-02-15

    The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10{sup 6} cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P < 0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a

  3. Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

    Science.gov (United States)

    Dinicola, Simona; Fabrizi, Gianmarco; Masiello, Maria Grazia; Proietti, Sara; Palombo, Alessandro; Minini, Mirko; Harrath, Abdel Halim; Alwasel, Saleh H; Ricci, Giulia; Catizone, Angela; Cucina, Alessandra; Bizzarri, Mariano

    2016-07-01

    Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.

  4. De novo DNA Methyltransferases Dnmt3a and Dnmt3b regulate the onset of Igκ light chain rearrangement during early B-cell development.

    Science.gov (United States)

    Manoharan, Anand; Du Roure, Camille; Rolink, Antonius G; Matthias, Patrick

    2015-08-01

    Immunoglobulin genes V(D)J rearrangement during early lymphopoiesis is a critical process involving sequential recombination of the heavy and light chain loci. A number of transcription factors act together with temporally activated recombinases and chromatin accessibility changes to regulate this complex process. Here, we deleted the de novo DNA methyltransferases Dnmt3a and Dnmt3b in early B cells of conditionally targeted mice, and monitored the process of V(D)J recombination. Dnmt3a and Dnmt3b deletion resulted in precocious recombination of the immunoglobulin κ light chain without impairing the differentiation of mature B cells or overall B-cell development. Ex vivo culture of IL-7 restricted early B-cell progenitors lacking Dnmt3a and Dnmt3b showed precocious Vκ-Jκ rearrangements that are limited to the proximal Vκ genes. Furthermore, B-cell progenitors deficient in Dnmt3a and Dnmt3b showed elevated levels of germline transcripts at the proximal Vκ genes, alterations in methylation patterns at Igκ enhancer sites and increased expression of the transcription factor E2A. Our data suggest that Dnmt3a and Dnmt3b are critical to regulate the onset of Igκ light chain rearrangement during early B-cell development.

  5. Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis.

    Science.gov (United States)

    Sá, João V; Kleiderman, Susanne; Brito, Catarina; Sonnewald, Ursula; Leist, Marcel; Teixeira, Ana P; Alves, Paula M

    2017-01-01

    Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-(13)C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained (13)C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis ((13)C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.

  6. Alectinib for choroidal metastasis in a patient with crizotinib-resistant ALK rearranged positive non-small cell lung cancer.

    Science.gov (United States)

    Okuma, Yusuke; Tanaka, Yuichiro; Kamei, Tina; Hosomi, Yukio; Okamura, Tatsuru

    2015-01-01

    Choroidal metastasis is rare in cancer patients. Small molecules of molecular targeted agents for lung cancer with actionable mutations were reported to be palliated for symptoms caused by choroidal metastasis. Visual disturbance by choroidal metastasis significantly decreases quality of life during the patient's remaining lifespan; therefore, radiotherapy or laser photocoagulation is proposed with consensus. However, improvement in survival with matched molecular targeted agents for oncogenic driver mutations reminds us to also be concerned with late treatment toxicities. A 30-year-old female patient previously treated with crizotinib harboring ALK rearranged non-small cell lung cancer complained of visual disturbance, fever, and bone pains undergoing anti-PD-1 antibody treatment. A decreased proportion of ALK fusion was demonstrated by fluorescence in situ hybridization in liver metastasis compared to the primary site in a chemo-naïve state. She was diagnosed with low vision, choroidal metastasis and retinal detachment. Therefore, she started alectinib treatment and both her ocular and systemic symptoms were palliated in a week. Later, she temporarily discontinued alectinib because of skin rash although the choroidal metastasis and retinal detachment resolved and she regained low vision completely at 2 weeks. She obtained partial response with alectinib for more than 5 months after recovering from skin rash.

  7. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    Science.gov (United States)

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

  8. : a database of ciliate genome rearrangements.

    Science.gov (United States)

    Burns, Jonathan; Kukushkin, Denys; Lindblad, Kelsi; Chen, Xiao; Jonoska, Nataša; Landweber, Laura F

    2016-01-01

    Ciliated protists exhibit nuclear dimorphism through the presence of somatic macronuclei (MAC) and germline micronuclei (MIC). In some ciliates, DNA from precursor segments in the MIC genome rearranges to form transcriptionally active genes in the mature MAC genome, making these ciliates model organisms to study the process of somatic genome rearrangement. Similar broad scale, somatic rearrangement events occur in many eukaryotic cells and tumors. The (http://oxytricha.princeton.edu/mds_ies_db) is a database of genome recombination and rearrangement annotations, and it provides tools for visualization and comparative analysis of precursor and product genomes. The database currently contains annotations for two completely sequenced ciliate genomes: Oxytricha trifallax and Tetrahymena thermophila.

  9. Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells.

    Directory of Open Access Journals (Sweden)

    Nurdan Ozkucur

    rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.

  10. Clinicopathological study of gene rearrangement and immunohistochemical pattern of primary intracranial diffuse large B-cell lymphomas

    Directory of Open Access Journals (Sweden)

    Han X

    2013-11-01

    Full Text Available We studied the clinicopathological and imaging characteristics of primary intracranial diffuse large B-cell lymphomas (PIC-DLBCL. Imaging, histopathological findings, and immunohistochemical staining characteristics were analyzed, and the immunoglobulin heavy and light chain gene rearrangement of 25 PIC-DLBCL cases was examined. MicroRNA was extracted from 10 cases each of PIC-DLBCL, extracerebral germinal center DLBCL (GC-DLBCL, and extracerebral non-GC-DLBCL (NGC-DLBCL; we conducted chip hybridization and comparatively analyzed the difference among the three. PIC-DLBCLs typically involved no less than two cerebral lobes (10/25; the frontal lobe was affected most often (6/25. Target-shaped structures were observed in all PIC-DLBCLs due to the proliferation of centroblast-like large lymphocytes surrounding the vessels. There was strong and diffuse immunostaining for CD20 and CD79a, and negative immunostaining for CD3, CD5, CD23, and cyclin D1 for all PIC-DLBCLs. The percentage of cells with nuclear positivity for anti-Ki67 antibody ranged 50-90% (mean, 80%. Three, 19, and 22 PIC-DLBCLs were CD10-, Bcl-6-, and melanoma ubiquitous mutated 1-positive, respectively. Twenty-four PIC-DLBCLs were B-cell monoclonal. MicroRNA hybridization showed that 788 PIC-DLBCL microRNAs/segments increased to at least twice of that of NGC-DLBCLs, and 401 PIC-DLBCL microRNAs/segments declined to less than half of that of NGC-DLBCLs. Six hundred and eleven PIC-DLBCL microRNAs/segments increased to at least twice that of GC-DLBCLs, and 229 PIC-DLBCL microRNAs/segments declined to less than half of that in GC-DLBCLs. PIC-DLBCL typically affected multiple sites, tended to occur in older men, arose from activated B cells, had high B-cell monoclonality; its microRNA expression differed from that of NGC-DLBCL and GC-DLBCL.

  11. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells.

    Science.gov (United States)

    Chiarle, Roberto; Zhang, Yu; Frock, Richard L; Lewis, Susanna M; Molinie, Benoit; Ho, Yu-Jui; Myers, Darienne R; Choi, Vivian W; Compagno, Mara; Malkin, Daniel J; Neuberg, Donna; Monti, Stefano; Giallourakis, Cosmas C; Gostissa, Monica; Alt, Frederick W

    2011-09-30

    Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

  12. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  13. The structure, rearrangement, and ontogenic expression of DB and JB gene segments of the Mexican axolotl T-cell antigen receptor beta chain (TCRB).

    Science.gov (United States)

    Kerfourn, F; Charlemagne, J; Fellah, J S

    1996-01-01

    We sequenced a total of 189 independent rearrangements in which the VB7.1 element is associated with CB1 (99 clones) or CB2 (90 clones) isotypes of the T-cell receptor (TCR) beta chain in the Mexican axolotl. Three stages of development were analyzed: 2.5 months, 10 months, and 25 months. Three JB1 segments were associated with the VB-CB1 rearrangements and six JB2 segments with VB-CB2. As in other vertebrates, some amino acid positions were conserved in all Jbetas (e. g., Phe-108, Gly-109, Gly-111, Thr-112, and Val-116). Two 11 nucleotides DB-like sequences, differed by one (A or T) central residue and could be productively read in the three putative reading frames. Most of the DB1 and JB1 segments were in the VB-CB1 clones, and most of the DB2 and JB2 segments were in the VB-CB2 clones, suggesting that the TCRB locus is organized into independent DB-JB-CB clusters that used the same collection of VB segments. About 40% of the beta-chain VDJ junctions in 2.5-month-old larvae had N nucleotides, compared with about 73% in 10 - 25-month old animals. The beta-chain VDJ junctions had about 30% of defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs, and the large genome in this urodele. Many of the axolotl CDRbeta3 sequences deduced for in frame VDJ rearrangements are the same in animals of different origins. Such redundancy could be a statistical effect due to the small number of thymocytes in the developing axolotl, rather than to some bias due to junctional preferences.

  14. Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis

    OpenAIRE

    Gascoigne, Karen E; Cheeseman, Iain M.

    2013-01-01

    Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a “dicentric” chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cel...

  15. Sample preparation by cell guiding using negative dielectrophoresis

    DEFF Research Database (Denmark)

    Christensen, Troels Balmer; Pedersen, Christian Møller; Bang, Dang Duong;

    2007-01-01

    In this study, we present a microsystem designed for performing and testing dielectrophoretic (DEP) guiding of biological cells. Baker's yeast (Saccharomyces cerevisiae) is used as a model organism to study cell guiding in the system. The guiding efficiency as a function of flowrate is investigated...... and a decreased efficiency with increased flowrate is observed. In addition, the DEP behaviour of the yeast cells at different medium conductivities and applied frequencies is investigated. The chip is easily fabricated in a two-step process: Standard UV lithography techniques are used for electrode fabrication...

  16. 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...

  17. Characterization of clonal immunoglobulin heavy (IGH) V-D-J gene rearrangements and the complementarity-determining region in South Indian patients with precursor B-cell acute lymphoblastic leukemia

    Science.gov (United States)

    Rajkumar, Thangarajan; Rajalekshmy, Kamalalayam Raghavan; Nancy, Nirmala Karunakaran

    2017-01-01

    Background This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identified age-related predominance in VDJ rearrangements. Methods IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. Results Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A somatic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. Conclusion The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predominantly used in precursor-B-ALL. However, the IGH gene rearrangements in precursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.

  18. Severe acute interstitial lung disease in a patient with anaplastic lymphoma kinase rearrangement-positive non-small cell lung cancer treated with alectinib.

    Science.gov (United States)

    Yamamoto, Yuzo; Okamoto, Isamu; Otsubo, Kohei; Iwama, Eiji; Hamada, Naoki; Harada, Taishi; Takayama, Koichi; Nakanishi, Yoichi

    2015-10-01

    Alectinib, the second generation anaplastic lymphoma kinase (ALK) inhibitor, has significant potency in patients with ALK rearrangement positive non-small cell lung cancer (NSCLC), and its toxicity is generally well tolerable. We report a patient who developed severe acute interstitial lung disease after alectinib treatment. An 86-year-old woman with stage IV lung adenocarcinoma positive for rearrangement of ALK gene was treated with alectinib. On the 215th day after initiation of alectinib administration, she was admitted to our hospital with the symptom of progressive dyspnea. Computed tomography (CT) revealed diffuse ground glass opacities and consolidations in both lungs, and analysis of bronchoalveolar lavage fluid revealed pronounced lymphocytosis. There was no evidence of infection or other specific causes of her condition, and she was therefore diagnosed with interstitial lung disease induced by alectinib. Her CT findings and respiratory condition improved after steroid pulse therapy. As far as we are aware, this is the first reported case of alectinib-induced severe interstitial lung disease (ILD). We should be aware of the possibility of such a severe adverse event and should therefore carefully monitor patients treated with this drug.

  19. Rearrangements of Cycloalkenyl Aryl Ethers

    Directory of Open Access Journals (Sweden)

    Mercedesz Törincsi

    2016-04-01

    Full Text Available Rearrangement reactions of cycloalkenyl phenol and naphthyl ethers and the acid-catalyzed cyclization of the resulting product were investigated. Claisen rearrangement afforded 2-substituted phenol and naphthol derivatives. Combined Claisen and Cope rearrangement resulted in the formation of 4-substituted phenol and naphthol derivatives. In the case of cycloocthylphenyl ether the consecutive Claisen and Cope rearrangements were followed by an alkyl migration. The mechanism of this novel rearrangement reaction is also discussed.

  20. New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

    Science.gov (United States)

    Vedyaykin, Alexey D; Vishnyakov, Innokentii E; Polinovskaya, Vasilisa S; Khodorkovskii, Mikhail A; Sabantsev, Anton V

    2016-06-01

    FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction.

  1. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

    Science.gov (United States)

    Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

    2013-01-01

    Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

  2. T cell receptor gene rearrangement of Peripheral blood mononuclear cells from patients with chronic hepatitis B%慢性乙型肝炎患者外周血T细胞受体基因重组的意义

    Institute of Scientific and Technical Information of China (English)

    郭毅; 吴辉; 李十月; 燕虹

    2000-01-01

    目的 通过对慢性乙型肝炎患者外周血T细胞受体基因β链(T ceIl receptor gene Beta chain,TCRβ)重组与血清HBeAg,抗-HBe和丙氨酸转氨酶(ALT)平行检测,以探讨优势T细胞克隆在病程中的作用.方法 运用Southern杂交检测85例慢性乙型肝炎患者外周血单个核细胞DNA.结果 85例患者TCRβ重组阳性率为44.7%,大多数具有EcoRI或/和Hindm重组带.横断面比较及对19例患者的随访表明:TCRβ基因重组的检出与HBV的清除和细胞毒作用有关.结论 TCRβ基因重组似可作为评价本病特异性免疫应答及肝脏损伤的一个指标.%Objective T cell receptor gene(TCR)rearrangement of peripheral blood mononuclear cells from patients with chronic hepatitis B was detected simultaneously with serum HBeAg,anti-HBe and alanine aminotransferase(ALT) to examine the role of dominant T cell clones.Methods Southern hy-bridization was used to detect DNA from peripheral blood mononuclear cells in 85 patients with chronic he-patitis B.ResuIts The positive rate of TCR rearrangement was 44.7%in 85 patients with chronic hepati-tis B.Most of them demonstrated two kinds of rearrangement bands of DNA fragment.Cross-sectional study indicated there was no significant difference in positive rate of HBeAg between TCR positive group and TCR negative group while there was significant difference in ALT between the two groups.Follow-up of 19 patients with chronic hepatitis B suggested that appearance of the TCR rearrangement was associated with cytotoxicity to liver and clearance of hepatitis B virus.Conclusion The detection of TCR rearrange-ment is useful in the evaluation of disease progression and immune response.

  3. Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Bürgin-Maunder, Corinna S; Brooks, Peter R; Russell, Fraser D

    2013-11-08

    Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  4. Omega-3 Fatty Acids Modulate Weibel-Palade Body Degranulation and Actin Cytoskeleton Rearrangement in PMA-Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Corinna S. Bürgin-Maunder

    2013-11-01

    Full Text Available Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF in cytoplasmic Weibel-Palade bodies (WPBs. We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs. HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA, and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

  5. Piracy of decay-accelerating factor (CD55) signal transduction by the diffusely adhering strain Escherichia coli C1845 promotes cytoskeletal F-actin rearrangements in cultured human intestinal INT407 cells.

    Science.gov (United States)

    Peiffer, I; Servin, A L; Bernet-Camard, M F

    1998-09-01

    Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cgamma, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+ inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

  6. Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study

    DEFF Research Database (Denmark)

    Mann, Georg; Attarbaschi, Andishe; Schrappe, Martin;

    2010-01-01

    To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL(+)), we compared the outcome of MLL(+) patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376...

  7. Multicenter Phase II Study of Whole-Body and Intracranial Activity With Ceritinib in Patients With ALK-Rearranged Non-Small-Cell Lung Cancer Previously Treated With Chemotherapy and Crizotinib : Results From ASCEND-2

    NARCIS (Netherlands)

    Crino, Lucio; Ahn, Myung-Ju; De Marinis, Filippo; Groen, Harry J. M.; Wakelee, Heather; Hida, Toyoaki; Mok, Tony; Spigel, David; Felip, Enriqueta; Nishio, Makoto; Scagliotti, Giorgio; Branle, Fabrice; Emeremni, Chetachi; Quadrigli, Massimiliano; Zhang, Jie; Shaw, Alice T.

    2016-01-01

    Purpose Phase I data (ASCEND-1) showed ceritinib efficacy in patients with ALK-rearranged non-small-cell lung cancer (NSCLC), regardless of brain metastases status and with or without prior therapy with an inhibitor of the ALK protein. Data are presented from a phase II trial (ASCEND-2) in which cer

  8. Environmental and chemotherapeutic agents induce breakage at genes involved in leukemia-causing gene rearrangements in human hematopoietic stem/progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Thys, Ryan G., E-mail: rthys@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016 (United States); Lehman, Christine E., E-mail: clehman@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016 (United States); Pierce, Levi C.T., E-mail: Levipierce@gmail.com [Human Longevity, Inc., San Diego, California 92121 (United States); Wang, Yuh-Hwa, E-mail: yw4b@virginia.edu [Department of Biochemistry and Molecular Genetics, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0733 (United States)

    2015-09-15

    Highlights: • Environmental/chemotherapeutic agents cause DNA breakage in MLL and CBFB in HSPCs. • Diethylnitrosamine-induced DNA breakage at MLL and CBFB shown for the first time. • Chemical-induced DNA breakage occurs at topoisomerase II cleavage sites. • Chemical-induced DNA breaks display a pattern similar to those in leukemia patients. • Long-term exposures suggested to generate DNA breakage at leukemia-related genes. - Abstract: Hematopoietic stem and progenitor cells (HSPCs) give rise to all of the cells that make up the hematopoietic system in the human body, making their stability and resilience especially important. Damage to these cells can severely impact cell development and has the potential to cause diseases, such as leukemia. Leukemia-causing chromosomal rearrangements have largely been studied in the context of radiation exposure and are formed by a multi-step process, including an initial DNA breakage and fusion of the free DNA ends. However, the mechanism for DNA breakage in patients without previous radiation exposure is unclear. Here, we investigate the role of non-cytotoxic levels of environmental factors, benzene, and diethylnitrosamine (DEN), and chemotherapeutic agents, etoposide, and doxorubicin, in generating DNA breakage at the patient breakpoint hotspots of the MLL and CBFB genes in human HSPCs. These conditions represent exposure to chemicals encountered daily or residual doses from chemotherapeutic drugs. Exposure of HSPCs to non-cytotoxic levels of environmental chemicals or chemotherapeutic agents causes DNA breakage at preferential sites in the human genome, including the leukemia-related genes MLL and CBFB. Though benzene, etoposide, and doxorubicin have previously been linked to leukemia formation, this is the first study to demonstrate a role for DEN in the generation of DNA breakage at leukemia-specific sites. These chemical-induced DNA breakpoints coincide with sites of predicted topoisomerase II cleavage. The

  9. Prognostic impact of concurrent MYC and BCL6 rearrangements and expression in de novo diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Ye, Qing; Xu-Monette, Zijun Y; Tzankov, Alexandar;

    2016-01-01

    and BCL6 in 898 patients with de novo diffuse large B-cell lymphoma treated with standard chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab). Neither BCL6 translocation alone (more frequent in activated B-cell like diffuse large B-cell lymphoma) nor in combination...

  10. Recurrent DNA inversion rearrangements in the human genome

    DEFF Research Database (Denmark)

    Flores, Margarita; Morales, Lucía; Gonzaga-Jauregui, Claudia

    2007-01-01

    Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome...... on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic...... cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard...

  11. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Wu

    Full Text Available BACKGROUND: Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR, fluorescence in Situ hybridization (FISH and Immunohistochemical (IHC stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. RESULTS: Thirteen of the 312 patients (4.17% had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%, but very good specificity (99.32%. IHC stain had better sensitivity (91.67% than FISH, but lower specificity (79.52%, when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product, were also have high expression of ALK protein (IHC3+, and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80% of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%. Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3% was the most common type in Chinese population, while variant 1 (28/37, 75.7% was most common in Caucasian. CONCLUSIONS: Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

  12. Standard guide for general design considerations for hot cell equipment

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2008-01-01

    1.1 Intent: 1.1.1 The intent of this guide is to provide general design and operating considerations for the safe and dependable operation of remotely operated hot cell equipment. Hot cell equipment is hardware used to handle, process, or analyze nuclear or radioactive material in a shielded room. The equipment is placed behind radiation shield walls and cannot be directly accessed by the operators or by maintenance personnel because of the radiation exposure hazards. Therefore, the equipment is operated remotely, either with or without the aid of viewing. 1.1.2 This guide may apply to equipment in other radioactive remotely operated facilities such as suited entry repair areas, canyons or caves, but does not apply to equipment used in commercial power reactors. 1.1.3 This guide does not apply to equipment used in gloveboxes. 1.2 Applicability: 1.2.1 This guide is intended for persons who are tasked with the planning, design, procurement, fabrication, installation, or testing of equipment used in rem...

  13. Rearrangements in ground and excited states

    CERN Document Server

    de Mayo, Paul

    1980-01-01

    Rearrangements in Ground and Excited States, Volume 2 covers essays on the theoretical approach of rearrangements; the rearrangements involving boron; and the molecular rearrangements of organosilicon compounds. The book also includes essays on the polytopal rearrangement at phosphorus; the rearrangement in coordination complexes; and the reversible thermal intramolecular rearrangements of metal carbonyls. Chemists and people involved in the study of rearrangements will find the book invaluable.

  14. Rearrangements in ground and excited states

    CERN Document Server

    de Mayo, Paul

    1980-01-01

    Rearrangements in Ground and Excited States, Volume 3 presents essays on the chemical generation of excited states; the cis-trans isomerization of olefins; and the photochemical rearrangements in trienes. The book also includes essays on the zimmerman rearrangements; the photochemical rearrangements of enones; the photochemical rearrangements of conjugated cyclic dienones; and the rearrangements of the benzene ring. Essays on the photo rearrangements via biradicals of simple carbonyl compounds; the photochemical rearrangements involving three-membered rings or five-membered ring heterocycles;

  15. An atypical human induced pluripotent stem cell line with a complex, stable, and balanced genomic rearrangement including a large de novo 1q uniparental disomy.

    Science.gov (United States)

    Steichen, Clara; Maluenda, Jérôme; Tosca, Lucie; Luce, Eléanor; Pineau, Dominique; Dianat, Noushin; Hannoun, Zara; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne

    2015-03-01

    Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts.

  16. Nano-guided cell networks as conveyors of molecular communication.

    Science.gov (United States)

    Terrell, Jessica L; Wu, Hsuan-Chen; Tsao, Chen-Yu; Barber, Nathan B; Servinsky, Matthew D; Payne, Gregory F; Bentley, William E

    2015-01-01

    Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and 'binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a 'bio-litmus' in a manner read by simple optical means.

  17. Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence.

    Science.gov (United States)

    Rueda, Cristina; Cuenca-Estrella, Manuel; Zaragoza, Oscar

    2014-01-01

    In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme β-1,3-d-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, β-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.

  18. N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements

    NARCIS (Netherlands)

    J.G. Noordzij; N.S. Verkaik (Nicole); N.G. Hartwig (Nico); R. de Groot (Ronald); D.C. van Gent (Dik); J.J.M. van Dongen (Jacques)

    2000-01-01

    textabstractThe proteins encoded by RAG1 and RAG2 can initiate gene recombination by site-specific cleavage of DNA in immunoglobulin and T-cell receptor (TCR) loci. We identified a new homozygous RAG1 gene mutation (631delT) that leads to a premature stop codon in the 5

  19. IMPre: an accurate and efficient software for prediction of T- and B-cell receptor germline genes and alleles from rearranged repertoire data

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2016-11-01

    Full Text Available Large-scale study of the properties of T-cell receptor (TCR and B-cell receptor (BCR repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(DiversityJoining V(DJ germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed Immune Germline Prediction (IMPre, a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, Seed_Clust, for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB and immunoglobulin heavy chain (IGH, and then tested it on additional human samples. Accuracy of 97.7%, 100%, 92.9% and 100% was obtained for TRBV, TRBJ, IGHV and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre.

  20. High levels of nuclear MYC protein predict the presence of MYC rearrangement in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Green, Tina Marie; Nielsen, Ole; de Stricker, Karin;

    2012-01-01

    , and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found...... a significant positive correlation between levels of MYC transcripts and MYC tumor cells; however, QRT-PCR is not readily applicable as a screening tool. Immunohistochemically, all tumors showed a nuclear staining pattern that was simple to evaluate. The percentage of MYC lymphoma cells correlated closely...... with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry...

  1. Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

    Directory of Open Access Journals (Sweden)

    Michelle S Itano

    2014-08-01

    Full Text Available Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (~80 nm on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to four hours. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal nanodomain structure, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of < 30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

  2. Diagnostic and therapeutic issues for patients with advanced non‑small cell lung cancer harboring anaplastic lymphoma kinase rearrangement: European vs. US perspective (review).

    Science.gov (United States)

    Di Maio, Massimo; De Marinis, Filippo; Hirsch, Fred R; Gridelli, Cesare

    2014-08-01

    The recent availability of crizotinib in clinical practice, for the treatment of patients with advanced non-small cell lung cancer (NSCLC) selected by the presence of anaplastic lymphoma kinase (ALK) rearrangement, has relevant implications for both the diagnostic phase and the treatment choices. In the United States, crizotinib was approved by the Food and Drug Administration (FDA) in 2011 for patients with ALK positivity detected by FDA-approved companion diagnostic test. As of January, 2014, the only FDA-approved diagnostic test is Vysis ALK Break-Apart FISH Probe Kit. In Europe, European Medicines Agency (EMA) approved crizotinib for ALK-positive patients in 2012, without specifying the type of test used for determining the positivity. FISH remains the reference technique for ALK determination, but, if fully validated, immunohistochemistry could challenge the current ALK screening practice. Given the robust evidence of activity of crizotinib in ALK-positive patients both pretreated and chemotherapy-naïve, and the favourable tolerability profile of the drug, many oncologists would prefer to administer the drug as early as possible. This is technically feasible in the United States, where crizotinib was approved well before the availability of the results of the randomized phase III trial comparing the drug with standard second-line chemotherapy, and the use of crizotinib in ALK-positive patients is not restricted to a specific line of treatment. On the contrary, in Europe, differently from the FDA decision, crizotinib cannot be used in chemotherapy-naïve patients. In both realities, a deeper knowledge of mechanisms of resistance, the role of repeated biopsies, the treatment strategy for patients experiencing disease progression with crizotinib, the choice of the best chemotherapy regimen are challenging topics for the management of ALK-positive patients in clinical practice.

  3. Standard guide for hot cell specialized support equipment and tools

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 Intent: 1.1.1 This guide presents practices and guidelines for the design and implementation of equipment and tools to assist assembly, disassembly, alignment, fastening, maintenance, or general handling of equipment in a hot cell. Operating in a remote hot cell environment significantly increases the difficulty and time required to perform a task compared to completing a similar task directly by hand. Successful specialized support equipment and tools minimize the required effort, reduce risks, and increase operating efficiencies. 1.2 Applicability: 1.2.1 This guide may apply to the design of specialized support equipment and tools anywhere it is remotely operated, maintained, and viewed through shielding windows or by other remote viewing systems. 1.2.2 Consideration should be given to the need for specialized support equipment and tools early in the design process. 1.2.3 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conv...

  4. GVHD (Graft-Versus-Host Disease): A Guide for Patients and Families After Stem Cell Transplant

    Science.gov (United States)

    ... Disease): A guide for patients and families after stem cell transplant The immune system is the body's tool ... and attacking them. When you receive a donor's stem cells (the “graft”), the stem cells recreate the donor's ...

  5. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    Science.gov (United States)

    Wang, Jinghui; Cai, Yiran; Dong, Yujie; Nong, Jingying; Zhou, Lijuan; Liu, Guimei; Su, Dan; Li, Xi; Wu, Shafei; Chen, Xuejing; Qin, Na; Zeng, Xuan; Zhang, Haiqing; Zhang, Zongde; Zhang, Shucai

    2014-01-01

    EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  6. Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR.

    Directory of Open Access Journals (Sweden)

    Jinghui Wang

    Full Text Available EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH, Ventana immunohistochemistry (IHC, and reverse transcriptase polymerase chain reaction (RT-PCR in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7% harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients' targeted therapy using crizotinib.

  7. Characterization of the T-cell receptor gamma chain gene rearrangements as an adjunct tool in the diagnosis of T-cell lymphomas in the gastrointestinal tract of cats.

    Science.gov (United States)

    Gress, Verena; Wolfesberger, Birgitt; Fuchs-Baumgartinger, Andrea; Nedorost, Nora; Saalmüller, Armin; Schwendenwein, Ilse; Rütgen, Barbara C; Hammer, Sabine E

    2016-08-01

    Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.

  8. Cytomegalovirus (CMV) Infection: A Guide for Patients and Families After Stem Cell Transplant

    Science.gov (United States)

    ... Infection: A Guide for Patients and Families after Stem Cell Transplant What is cytomegalovirus (CMV)? Cytomegalovirus (CMV), a ... weakened by medicines that you must take after stem cell transplant and by the transplant itself. Your body ...

  9. Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications

    Directory of Open Access Journals (Sweden)

    Kazi M. Zakir Hossain

    2015-07-01

    Full Text Available Tubular scaffolds with aligned polylactic acid (PLA fibres were fabricated for cell guide applications by immersing rolled PLA fibre mats into a polyvinyl acetate (PVAc solution to bind the mats. The PVAc solution was also mixed with up to 30 wt % β-tricalcium phosphate (β-TCP content. Cross-sectional images of the scaffold materials obtained via scanning electron microscopy (SEM revealed the aligned fibre morphology along with a significant number of voids in between the bundles of fibres. The addition of β-TCP into the scaffolds played an important role in increasing the void content from 17.1% to 25.3% for the 30 wt % β-TCP loading, which was measured via micro-CT (µCT analysis. Furthermore, µCT analyses revealed the distribution of aggregated β-TCP particles in between the various PLA fibre layers of the scaffold. The compressive modulus properties of the scaffolds increased from 66 MPa to 83 MPa and the compressive strength properties decreased from 67 MPa to 41 MPa for the 30 wt % β-TCP content scaffold. The scaffolds produced were observed to change into a soft and flexible form which demonstrated shape recovery properties after immersion in phosphate buffered saline (PBS media at 37 °C for 24 h. The cytocompatibility studies (using MG-63 human osteosarcoma cell line revealed preferential cell proliferation along the longitudinal direction of the fibres as compared to the control tissue culture plastic. The manufacturing process highlighted above reveals a simple process for inducing controlled cell alignment and varying porosity features within tubular scaffolds for potential tissue engineering applications.

  10. THE RELATIONSHIP BETWEEN NON-HODGKIN'S LYMPHOMA AND THE GENE REARRANGEMENT

    Institute of Scientific and Technical Information of China (English)

    Guo Sutang; Liu Yongchang; Sun Junning

    1998-01-01

    Objective:To investigate the pattern of clonal rearrangement of immunoglobulin heavy chain gene (IGH) and T-cell receptor γ gene (TCRγ) of NonHodgkin's lymphoma (NHL). Methods: Bone marrow smears of 211 patients of NHL were detected by PCR, the rearranged IGH and TCRγ gene was amplified using oligonucleotide primers. Results: The clonal rearrangement of IGH gene was detectable in 51.2%(108/211); the clonal rearrangement of TCRγ gene was detectable in 21.3% (45/211); both IGH and TCRγwas detectable in 5.7% (12/211);no clonal rearrangement in 21.8% (46/211). And compared clonal gene rearrangement with pathological type and primary site of tumor. Ten patients of NHL were investigated serially. 5/10 patients still had clonal gene rearrangement at clinical complete remission. Conclusion: It demonstrated that this assay may be useful in monitoring the minimal residual disease (MRD) and in evaluating effectiveness of therapy.

  11. Lack of nonfunctional B-cell receptor rearrangements in a patient with normal B cell numbers despite partial RAG1 deficiency and atypical SCID/Omenn syndrome

    DEFF Research Database (Denmark)

    Ohm-Laursen, Line; Nielsen, Christian; Fisker, Niels

    2008-01-01

    and the patient had eosinophilia. These presentations are consistent with atypical severe combined immunodeficiency (SCID)/Omenn Syndrome and the diagnosis was confirmed by demonstration of homozygosity for the R841W mutation in the catalytic core of RAG1. Comparison of the patient's immunoglobulin heavy chain...... chromosome 14. CONCLUSION: We hypothesize that the R841W mutation causes a malfunction of RAG1 that has differential outcome on V(D)J recombination in B and T cells, as the patient had normal B cell numbers but suffered severe alpha-beta T-cell immunodeficiency.......INTRODUCTION: A 2.5-month old boy presented with recurrent wheezing, protracted diarrhea, erythrodermia, and failure to thrive. METHODS AND RESULTS: Laboratory analysis showed lymphocytopenia with severely reduced T-cell numbers but normal numbers of B and NK cells. Serum IgE was increased...

  12. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    Science.gov (United States)

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  13. Increased frequency of {gamma}{delta} T cells in cerebrospinal fluid and peripheral blood of patients with multiple sclerosis: Reactivity, cytotoxicity, and T cell receptor V gene rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Stinissen, P.; Vandevyver, C.; Medaer, R. [Dr. L. Willems Institute, Diepenbeek (Belgium)] [and others

    1995-05-01

    Infiltrating {gamma}{delta} T cells are potentially involved in the central nervous system demyelination in multiple sclerosis (MS). To further study this hypothesis, we analyzed the frequency and functional properties of {gamma}{delta} T cells in peripheral blood (PB) and paired cerebrospinal fluid (CSF) of patients with MS and control subjects, including patients with other neurologic diseases (OND) and healthy individuals. The frequency analysis was performed under limiting dilution condition using rIL-2 and PHA. After PHA stimulation, a significantly increased frequency of {gamma}{delta} T cells was observed in PB and in CSF of MS patients as compared with PB and CSF of patients with OND. The frequency was represented equally in OND patients and normal individuals. Similarly, the IL-2-responsive {gamma}{delta} T cells occurred at a higher frequency in PB of MS than of control subjects. Forty-three percent of the {gamma}{delta} T cell clones isolates from PB and CSF of MS patients responded to heat shock protein (HSP70) but not HSP65, whereas only 2 of 30 control {gamma}{delta} T cell clones reacted to the HSP. The majority of the {gamma}{delta} T cell clones were able to induce non-MHC-restricted cytolysis of Daudi cells. All clones displayed a substantial reactivity to bacterial superantigens staphylococcal enterotoxin B and toxic shock syndrome toxin-1, irrespective of their {gamma}{delta} V gene usage. Furthermore, the {gamma}{delta} T cell clones expressed predominantly TCRDV2 and GV2 genes, whereas the clones derived from CSF of MS patients expressed either DV1 or DV2 genes. The obtained {gamma}{delta} clones, in general, represented rather heterogeneous clonal origins, even though a predominant clonal origin was found in a set of 10 {gamma}{delta} clones derived from one patient with MS. The present study provides new evidence supporting a possible role of {gamma}{delta} T cells in the secondary inflammatory processes in MS. 39 refs., 5 figs., 4 tabs.

  14. Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.

    Science.gov (United States)

    Feng, Jun-Feng; Liu, Jing; Zhang, Xiu-Zhen; Zhang, Lei; Jiang, Ji-Yao; Nolta, Jan; Zhao, Min

    2012-02-01

    Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, it is critical to investigate how human NSCs (hNSCs) respond to EF before any possible clinical attempt. Aiming to characterize the EF-stimulated and guided migration of hNSCs, we derived hNSCs from a well-established human embryonic stem cell line H9. Small applied DC EFs, as low as 16 mV/mm, induced significant directional migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode increased with the increase of field strength. (Rho-kinase) inhibitor Y27632 is used to enhance viability of stem cells and has previously been reported to inhibit EF-guided directional migration in induced pluripotent stem cells and neurons. However, its presence did not significantly affect the directionality of hNSC migration in an EF. Cytokine receptor [C-X-C chemokine receptor type 4 (CXCR4)] is important for chemotaxis of NSCs in the brain. The blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional migration. Applied EFs could potentially be further exploited to guide hNSCs to injured sites in the central nervous system to improve the outcome of various diseases.

  15. Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.

    Science.gov (United States)

    Gascoigne, Karen E; Cheeseman, Iain M

    2013-07-01

    Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

  16. Diagnostic significance of TCR gene clonal rearrangement analysis in early mycosis fungoides

    Institute of Scientific and Technical Information of China (English)

    Chen Xu; Chuan Wan; Lin Wang; Han-Jun Yang; Yuan Tang; Wei-Ping Liu

    2011-01-01

    Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, has various unspecific clinical and histological characteristics. Its eariy diagnosis is challenging. The application of T-cell receptor (TCR) gene clonal rearrangement to the diagnosis of MF has been widely studied. In this study, we used polymerase chain reaction (PCR) to investigate the diagnostic significance of detecting TCR-γ and -β gene clonal rearrangement in the eady diagnosis of mycosis fungoides. PCR for TCR-γ and TCR-β gene rearrangement was performed on 19 patients with suspected early MF, 6 with typical MF, and 6 with chronic dermatitis. Of the 19 patients with suspected eady MF, 13 had TCR-~ gene clonal rearrangement, whereas none had TCR-β gene clonal rearrangement. All patients with typical MF had TCR gene clonal rearrangement, in which 4 showed TCR-γ clonal rearrangement, 1 showed TCR-β gene clonal rearrangements, and 1 showed both. No patients with chronic dermatitis had TCR gene clonal rearrangement. These results indicate that TCR gene clonal rearrangement analysis is a useful tool in diagnosing early MF. TCR-γ gene is recommended to the routine analysis, whereas TCR-β gene has potential in combination toward intractable cases.

  17. Clues to pathogenesis of Waldenström macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance provided by analysis of immunoglobulin heavy chain gene rearrangement and clustering of B-cell receptors.

    Science.gov (United States)

    Varettoni, Marzia; Zibellini, Silvia; Capello, Daniela; Arcaini, Luca; Rossi, Davide; Pascutto, Cristiana; Rattotti, Sara; Mangiacavalli, Silvia; Pochintesta, Lara; Gotti, Manuel; Gaidano, Gianluca; Cazzola, Mario

    2013-11-01

    We characterized immunoglobulin heavy chain (IGH) gene rearrangements and searched for clusters of stereotyped B-cell receptors in 123 patients with Waldenström macroglobulinemia (WM; n = 59) or immunoglobulin M monoclonal gammopathy of undetermined significance (IgM-MGUS) (n = 64). A productive monoclonal IGHV-D-J rearrangement was obtained in 99/123 patients (80%). Immunoglobulin heavy chain variable (IGHV) genes were mutated in 94/99 patients (95%) with a median somatic hypermutation rate of 6.7% (2.1-14.5). Compared with the normal B-cell repertoire, patients with WM/IgM-MGUS showed an over-representation of the IGHV3 subgroup (83% vs. 55%, p < 0.0001) and an under-representation of IGHV1 (7% vs. 14%, p = 0.04) and IGHV4 (7% vs. 23%, p = 0.0001) subgroups. At the gene level, in WM/IgM-MGUS there was an over-representation of IGHV3-23 (24% vs. 12%, p = 0.0003), IGHV3-64 (3% vs. < 1%, p = 0.003), IGHV3-7 (12% vs. 4%, p = 0.0001) and IGHV3-74 (9% vs. 2%, p < 0.0001), while IGHV4-39 was never used (0 vs. 5%, p = 0.03). Intra-WM/IgM-MGUS search for HCDR3 similarity showed no association fulfilling criteria for stereotyped receptors. WM/IgM-MGUS sequences were unrelated to known chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL) or mantle cell lymphoma (MCL) subsets. In conclusion, the IGHV gene usage in WM and IgM-MGUS is remarkably biased as compared to the normal B-cell repertoire. WM and IgM-MGUS-specific HCDR3 clusters do not occur with a frequency detectable with currently available databases, not supporting a B-cell receptor-driven pathogenesis in WM and IgM-MGUS.

  18. Laser ablation of basal cell carcinomas guided by confocal microscopy

    Science.gov (United States)

    Sierra, Heidy; Cordova, Miguel; Nehal, Kishwer; Rossi, Anthony; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2016-02-01

    Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm2, thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

  19. Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

    Directory of Open Access Journals (Sweden)

    Manami Miyai

    Full Text Available Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01 in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB gene next generation sequencing (NGS to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3 rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF. Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133% even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB

  20. Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

    Science.gov (United States)

    Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-01-01

    Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS

  1. Significance of myc gene rearrangement and its correlation with prognosis in diffuse large B cell lymphoma%弥漫性大B细胞淋巴瘤中myc基因重排及其临床意义

    Institute of Scientific and Technical Information of China (English)

    张宏伟; 陈振文; 贺建霞; 郑玉萍; 韩维娥; 赵志强; 白玮; 王晋芬

    2013-01-01

    Objective To study the relationship between myc gene rearrangement and myc protein expression in diffuse large B cell lymphoma (DLBCL),and their correlation with prognosis.Methods One hundred and six cases of DLBCLs with follow-up data were analyzed using interphase fluorescence in situ hybridization (FISH) technique.Immunophenotyping analysis for CD20,CD3,myc,Mum-1,CD10,bcl-6 was also performed using EnVision immunohistochemistry.Results The percentages of tumor cells expressing myc,Mum-1,CD10 and bcl-6 were 70.8%,56.6%,21.7% and 26.4%,respectively.Twenty six cases(24.5%)were of GCB type and the rest(75.5%)were of non-GCB (non germinal center) type.The myc rearrangement was identified in 13 (12.3%) of 106 cases.13 cases showed to be of non-GCB type.There was no correlation between myc rearrangement and myc protein expression.DLBCLs (n =13) with myc rearrangement showed significantly poorer overall survival (OS) and progression free survival (PFS),with a median OS and PFS time of 4.7 and 3.2 months,respectively (for OS and PFS,P <0.001).Multivariate analysis using Cox proportional hazard model confirmed that myc rearrangement,ECOG performance status of 2-4,immunophenotyping subgroup and myc protein were independent factors affecting the prognosis and significantly associated with the survival.However,myc rearrangement was the strongest prognostic factor.Conclusions DLBCL with myc gene rearrangement is a subgroup of non-GCB DLBCL with poor outcome.It is an independent and useful factor for prognosis in DLBCL.Expression of myc is influenced by many factors and myc rearrangement may be one of these factors.%目的 探讨弥漫性大B细胞淋巴瘤(DLBCL)中myc基因重排和蛋白表达及其与预后的相关性.方法 采用免疫组织化学EnVision法检测106例DLBCL患者肿瘤组织中CD3、CD10、CD20、bcl-6、多发性骨髓瘤原癌基因1(Mum-1)和myc蛋白的表达情况,采用荧光原位杂交(FISH)技术检测myc基因重排.结果 106

  2. Gene conversion in human rearranged immunoglobulin genes.

    Science.gov (United States)

    Darlow, John M; Stott, David I

    2006-07-01

    Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.

  3. Immunoglobulin gene rearrangements and the pathogenesis of multiple myeloma

    NARCIS (Netherlands)

    Gonzalez, David; van der Burg, Mirjam; Garcia-Sanz, Ramon; Fenton, James A.; Langerak, Anton W.; Gonzalez, Marcos; van Dongen, Jacques J. M.; Miguel, Jesus F. San; Morgan, Gareth J.

    2007-01-01

    The ability to rearrange the germ-line DNA to generate antibody diversity is an essential prerequisite for the production of a functional repertoire. While this is essential to prevent infections, it also represents the "Achilles heal" of the B-cell lineage, occasionally leading to malignant transfo

  4. Effect of Interferon-γ on the Basal and the TNFα-Stimulated Secretion of CXCL8 in Thyroid Cancer Cell Lines Bearing Either the RET/PTC Rearrangement Or the BRAF V600e Mutation

    Directory of Open Access Journals (Sweden)

    Mario Rotondi

    2016-01-01

    Full Text Available CXCL8 displays several tumor-promoting effects. Targeting and/or lowering CXCL8 concentrations within the tumor microenvironment would produce a therapeutic benefit. Aim of this study was to test the effect of IFNγ on the basal and TNFα-stimulated secretion of CXCL8 in TCP-1 and BCPAP thyroid cancer cell lines (harboring RET/PTC rearrangement and BRAF V600e mutation, resp.. Cells were incubated with IFNγ (1, 10, 100, and 1000 U/mL alone or in combination with TNF-α (10 ng/mL for 24 hours. CXCL8 and CXCL10 concentrations were measured in the cell supernatants. IFNγ inhibited in a dose-dependent and significant manner both the basal (ANOVA F: 22.759; p<0.00001 and the TNFα-stimulated (ANOVA F: 15.309; p<0.00001 CXCL8 secretions in BCPAP but not in TPC-1 cells (NS. On the other hand, IFNγ and IFNγ + TNF-α induced a significant secretion of CXCL10 in both BCPAP (p<0.05 and TPC-1 (p<0.05 cells. Transwell migration assay showed that (i CXCL8 increased cell migration in both TPC-1 and BCPAP cells; (ii IFNγ significantly reduced the migration only of BCPAP cells; and (iii CXCL8 reverted the effect of IFNγ. These results constitute the first demonstration that IFNγ inhibits CXCL8 secretion and in turn the migration of a BRAF V600e mutated thyroid cell line.

  5. Effect of Interferon-γ on the Basal and the TNFα-Stimulated Secretion of CXCL8 in Thyroid Cancer Cell Lines Bearing Either the RET/PTC Rearrangement Or the BRAF V600e Mutation

    Science.gov (United States)

    Rotondi, Mario; Coperchini, Francesca; Awwad, Oriana; Di Buduo, Christian A.; Abbonante, Vittorio; Magri, Flavia; Balduini, Alessandra

    2016-01-01

    CXCL8 displays several tumor-promoting effects. Targeting and/or lowering CXCL8 concentrations within the tumor microenvironment would produce a therapeutic benefit. Aim of this study was to test the effect of IFNγ on the basal and TNFα-stimulated secretion of CXCL8 in TCP-1 and BCPAP thyroid cancer cell lines (harboring RET/PTC rearrangement and BRAF V600e mutation, resp.). Cells were incubated with IFNγ (1, 10, 100, and 1000 U/mL) alone or in combination with TNF-α (10 ng/mL) for 24 hours. CXCL8 and CXCL10 concentrations were measured in the cell supernatants. IFNγ inhibited in a dose-dependent and significant manner both the basal (ANOVA F: 22.759; p < 0.00001) and the TNFα-stimulated (ANOVA F: 15.309; p < 0.00001) CXCL8 secretions in BCPAP but not in TPC-1 cells (NS). On the other hand, IFNγ and IFNγ + TNF-α induced a significant secretion of CXCL10 in both BCPAP (p < 0.05) and TPC-1 (p < 0.05) cells. Transwell migration assay showed that (i) CXCL8 increased cell migration in both TPC-1 and BCPAP cells; (ii) IFNγ significantly reduced the migration only of BCPAP cells; and (iii) CXCL8 reverted the effect of IFNγ. These results constitute the first demonstration that IFNγ inhibits CXCL8 secretion and in turn the migration of a BRAF V600e mutated thyroid cell line. PMID:27555670

  6. Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.

    Science.gov (United States)

    Fu, Yanfang; Reyon, Deepak; Joung, J Keith

    2014-01-01

    CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.

  7. A reference guide to microbial cell surface hydrophobicity based on contact angles

    NARCIS (Netherlands)

    van der Mei, HC; Busscher, HJ; Bos, R.R.M.

    1998-01-01

    Acid-base interactions form the origin of the hydrophobicity of microbial cell-surfaces and can be quantitated from contact angle measurements on microbial lawns with water, formamide, methyleneiodide and/or alpha-bromonaphthalene. This review provides a reference guide to microbial cell surface hyd

  8. ETS rearrangements in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Mark A Rubin

    2012-01-01

    Prostate cancer is a clinically and molecularly heterogeneous disease.Understanding the biologic underpinning of prostate cancer is necessary to best determine how biology is associated with the risk of disease progression and how this understanding might provide insight into the development of novel therapeutic approaches.The focus of this review is on the recently identified common ETS and non-ETS gene rearrangements in prostate cancer.Although multiple molecular alterations have been detected in prostate cancer,a basic understanding of gene fusion prostate cancer should help explain the clinical and biologic diversity,providing a rationale for a molecular subclassification of the disease.

  9. Chromosomal rearrangements in Tourette syndrome

    DEFF Research Database (Denmark)

    Bertelsen, Birgitte; Debes, Nanette Mol; Hjermind, Lena E

    2013-01-01

    Tourette syndrome (TS) is a childhood-onset complex neurobiological disorder characterized by a combination of persistent motor and vocal tics and frequent presence of other neuropsychiatric comorbidities. TS shares the fate of other complex disorders, where the genetic etiology is largely unknown......, and identification of susceptibility genes through linkage and association studies has been complicated due to inherent difficulties such as no clear mode of inheritance, genetic heterogeneity, and apparently incomplete penetrance. Positional cloning through mapping of disease-related chromosome rearrangements has...

  10. Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

    Science.gov (United States)

    Bhargava, Ragini; Carson, Caree R; Lee, Gabriella; Stark, Jeremy M

    2017-01-24

    A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

  11. A new human natural killer leukemia cell line, IMC-1. A complex chromosomal rearrangement defined by spectral karyotyping: functional and cytogenetic characterization.

    Science.gov (United States)

    Chen, I-Ming; Whalen, Margaret; Bankhurst, Arthur; Sever, Cordelia E; Doshi, Rashmi; Hardekopf, David; Montgomery, Karen; Willman, Cheryl L

    2004-03-01

    A new human IL-2 dependent leukemic cell line with a natural killer (NK) cell phenotype, IMC-1, was established from an adult patient with aggressive NK cell leukemia. The IMC-1 cell line expresses the CD56, CD2, CD11a, CD38 and HLA-DR cell surface antigens, whereas the CD16 and CD8 antigens expressed on the primary leukemic blasts from which the cell line was derived were lost after 7 and 28 weeks of culture, respectively. The IMC-1 cell line displays functional NK cytotoxicity and lyses target cells in a non-MHC restricted, antibody-independent manner with equal or superior efficiency to freshly isolated NK cells. Cytogenetic analysis at presentation and after 55 weeks in culture revealed complex structural and numerical abnormalities, defined by classic G-banding and by spectral karyotyping (SKY). Three apparently intact copies of chromosome 8 occurred in the diagnostic bone marrow specimen; the cell line also contains three copies of chromosome 8 but each was structurally altered. The development and detailed characterization of this new NK leukemic cell line will facilitate biologic and functional studies of NK cells and chromosomal aberrations potentially important in leukemic transformation.

  12. Unidirectional cell crawling model guided by extracellular cues.

    Science.gov (United States)

    Wang, Zhanjiang; Geng, Yuxu

    2015-03-01

    Cell migration is a highly regulated and complex cellular process to maintain proper homeostasis for various biological processes. Extracellular environment was identified as the main affecting factors determining the direction of cell crawling. It was observed experimentally that the cell prefers migrating to the area with denser or stiffer array of microposts. In this article, an integrated unidirectional cell crawling model was developed to investigate the spatiotemporal dynamics of unidirectional cell migration, which incorporates the dominating intracellular biochemical processes, biomechanical processes and the properties of extracellular micropost arrays. The interpost spacing and the stiffness of microposts are taken into account, respectively, to study the mechanism of unidirectional cell locomotion and the guidance of extracellular influence cues on the direction of unidirectional cell crawling. The model can explain adequately the unidirectional crawling phenomena observed in experiments such as "spatiotaxis" and "durotaxis," which allows us to obtain further insights into cell migration.

  13. Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

    Science.gov (United States)

    Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L; Sand, Frederik; Heuckmann, Johannes M; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Gloeckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R; Savelyeva, Larissa; Watkins, Simon C; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H; Herrmann, Carl; O'Sullivan, Roderick J; Westermann, Frank; Thomas, Roman K; Fischer, Matthias

    2015-10-29

    Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

  14. Synthesis of a tricyclic lactam via Beckmann rearrangement and ring-rearrangement metathesis as key steps.

    Science.gov (United States)

    Kotha, Sambasivarao; Ravikumar, Ongolu; Majhi, Jadab

    2015-01-01

    A tricyclic lactam is reported in a four step synthesis sequence via Beckmann rearrangement and ring-rearrangement metathesis as key steps. Here, we used a simple starting material such as dicyclopentadiene.

  15. A quick guide to light microscopy in cell biology

    Science.gov (United States)

    Thorn, Kurt

    2016-01-01

    Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments. PMID:26768859

  16. Frequent TMPRSS2-ERG rearrangement in prostatic small cell carcinoma detected by fluorescence in situ hybridization: the superiority of fluorescence in situ hybridization over ERG immunohistochemistry.

    Science.gov (United States)

    Schelling, Lindsay A; Williamson, Sean R; Zhang, Shaobo; Yao, Jorge L; Wang, Mingsheng; Huang, Jiaoti; Montironi, Rodolfo; Lopez-Beltran, Antonio; Emerson, Robert E; Idrees, Muhammad T; Osunkoya, Adeboye O; Man, Yan-Gao; Maclennan, Gregory T; Baldridge, Lee Ann; Compérat, Eva; Cheng, Liang

    2013-10-01

    Small cell carcinoma of the prostate is both morphologically and immunohistochemically similar to small cell carcinoma of other organs such as the urinary bladder or lung. TMPRSS2-ERG gene fusion appears to be a highly specific alteration in prostatic carcinoma that is frequently shared by small cell carcinoma. In adenocarcinoma, immunohistochemistry for the ERG protein product has been reported to correlate well with the presence of the gene fusion, although in prostatic small cell carcinoma, this relationship is not completely understood. We evaluated 54 cases of small cell carcinoma of the prostate and compared TMPRSS2-ERG gene fusion status by fluorescence in situ hybridization (FISH) to immunohistochemical staining with antibody to ERG. Of 54 cases of prostatic small cell carcinoma, 26 (48%) were positive for TMPRSS2-ERG gene fusion by FISH and 12 (22%) showed overexpression of ERG protein by immunohistochemistry. Of the 26 cases positive by FISH, 11 were also positive for ERG protein by immunohistochemistry. One tumor was positive by immunohistochemistry but negative by FISH. Urinary bladder small cell carcinoma (n = 25) showed negative results by both methods; however, 2 of 14 small cell carcinomas of other organs (lung, head, and neck) showed positive immunohistochemistry but negative FISH. Positive staining for ERG by immunohistochemistry is present in a subset of prostatic small cell carcinomas and correlates with the presence of TMPRSS2-ERG gene fusion. Therefore, it may be useful in confirming prostatic origin when molecular testing is not accessible. However, sensitivity and specificity of ERG immunohistochemistry in small cell carcinoma are decreased compared to FISH.

  17. Gene Rearrangement Analysis of Orbital Lymphoid Infilktrating Disorders

    Institute of Scientific and Technical Information of China (English)

    JianghuaYan; ZhongyaoWu; 等

    2002-01-01

    Purpose:To determine whether the use of polymerae chain reaction for B-cell gene rearrangement in patients with orbital lymphoid infiltrate disorders could be useful in the diagnosis of lymphoma,especially,in differentiating benign lesion from malignant one.Methoids:In addition to clinical,pathological,and immunohistochemical evaluatons,48 cases of orbital lymphoid infiltrate disorders were examined for immunoglobulin heavy (IgH) gene rearrangement by means of PCR to amplify the FR3 region with formalin-fixed and paraffin-embedded tissues.Results:Gene rearrangement in the third frame-work of the IgH region was detected in specimens obtained from 15 cases of malignant lymphoma,4 of reactive lymphoid hyperplasia and 3 of orbital pseudotumor.All of these patients showed a discrete band (100bp) which reflected monoclonal proliferation of B lymphocytes.5 cases of malignant lymphoma,6 of reactive lymphoid hyperplasia and 15 of orbital pseudotumor did not show a discrete band on PCR.Conclusions:The FR3 region gene rearrangement of Ig heavy in patients with orbital lymphoid infilktrate disorders may be an additional diagnostic tool in differentiating benign from malignant lymphoid diseases and in offering a useful adjunct for diagnosis in difficult or unclear case.It is a reliable and practical method of gene diagnosis in orbital lymphoid infiltrate disorders and helps to identife the molecular mechanism of malignant lymphoma.Eye Science 2000;16:15-21.

  18. Gene Rearrangement Analysis of Orbital Lymphoid Infiltrating Disorders

    Institute of Scientific and Technical Information of China (English)

    Jianhua Yan; Zhongyao Wu; Shuqi Huang; Yongping Li

    2000-01-01

    Purpose: To determine whether the use of polymerase chain reaction for B-cell gene rearrangement in patients with orbital lymphoid infiltrate disorders could be useful in the diagnosis of lymphoma, especially, in differentiating benign lesion from malignant one. Methods: In addition to clinical, pathological, and immunohistochemical evaluations,48 cases of orbital lymphoid infiltrate disorders were examined for immunoglobulin heavy (IgH) gene rearrangement by means of PCR to amplify the FR3 region with formalin-fixed and paraffin-embedded tissues. Results: Gene rearrangement in the third frame-work of the IgH region was detected in specimens obtained from 15 cases of malignant lymphoma, 4 of reactive lymphoid hyperplasia and 3 of orbital pseudotumor. All of these patients showed a discrete band (100bp) which reflected monoclonal proliferation of B lymphocytes. 5 cases of malignant lymphoma, 6 of reactive lymphoid hyperplasia and 15 of orbital pseudotumor did not show a discrete band on PCR. Conclusions: The FR3 region gene rearrangement of Ig heavy in patients with orbital lymphoid infiltrate disorders may be an additional diagnostic tool in differentiating benign from malignant lymphoid diseases and in offering a useful adjunct for diagnosis in difficult or unclear cases. It is a reliable and practical method of gene diagnosis in orbital lymphoid infiltrate disorders and helps to identify the molecular mechanism of malignant lymphoma. Eye Science 2000; 16:15 ~ 21.

  19. Procuring Stationary Fuel Cells For CHP: A Guide for Federal Facility Decision Makers

    Energy Technology Data Exchange (ETDEWEB)

    Stinton, David P [ORNL; McGervey, Joseph [SRA International, Inc.; Curran, Scott [ORNL

    2011-11-01

    Federal agency leaders are expressing growing interest in using innovative fuel cell combined heat and power (CHP) technology at their sites, motivated by both executive branch sustainability targets and a desire to lead by example in the transition to a clean energy economy. Fuel cell CHP can deliver reliable electricity and heat with 70% to 85% efficiency. Implementing this technology can be a high efficiency, clean energy solution for agencies striving to meet ambitious sustainability requirements with limited budgets. Fuel cell CHP systems can use natural gas or renewable fuels, such as biogas. Procuring Stationary Fuel Cells for CHP: A Guide for Federal Facility Decision Makers presents an overview of the process for planning and implementing a fuel cell CHP project in a concise, step-by-step format. This guide is designed to help agency leaders turn their interest in fuel cell technology into successful installations. This guide concentrates on larger (100 kW and greater) fuel cell CHP systems and does not consider other fuel cell applications such as cars, forklifts, backup power supplies or small generators (<100 kW). Because fuel cell technologies are rapidly evolving and have high up front costs, their deployment poses unique challenges. The electrical and thermal output of the CHP system must be integrated with the building s energy systems. Innovative financing mechanisms allow agencies to make a make versus buy decision to maximize savings. This guide outlines methods that federal agencies may use to procure fuel cell CHP systems with little or no capital investment. Each agency and division, however, has its own set of procurement procedures. This guide was written as a starting point, and it defers to the reader s set of rules if differences exist. The fuel cell industry is maturing, and project developers are gaining experience in working with federal agencies. Technology improvements, cost reductions, and experienced project developers are making

  20. Rearrangements of organic peroxides and related processes.

    Science.gov (United States)

    Yaremenko, Ivan A; Vil', Vera A; Demchuk, Dmitry V; Terent'ev, Alexander O

    2016-01-01

    This review is the first to collate and summarize main data on named and unnamed rearrangement reactions of peroxides. It should be noted, that in the chemistry of peroxides two types of processes are considered under the term rearrangements. These are conventional rearrangements occurring with the retention of the molecular weight and transformations of one of the peroxide moieties after O-O-bond cleavage. Detailed information about the Baeyer-Villiger, Criegee, Hock, Kornblum-DeLaMare, Dakin, Elbs, Schenck, Smith, Wieland, and Story reactions is given. Unnamed rearrangements of organic peroxides and related processes are also analyzed. The rearrangements and related processes of important natural and synthetic peroxides are discussed separately.

  1. Rearrangements of organic peroxides and related processes

    Science.gov (United States)

    Yaremenko, Ivan A; Vil’, Vera A; Demchuk, Dmitry V

    2016-01-01

    Summary This review is the first to collate and summarize main data on named and unnamed rearrangement reactions of peroxides. It should be noted, that in the chemistry of peroxides two types of processes are considered under the term rearrangements. These are conventional rearrangements occurring with the retention of the molecular weight and transformations of one of the peroxide moieties after O–O-bond cleavage. Detailed information about the Baeyer−Villiger, Criegee, Hock, Kornblum−DeLaMare, Dakin, Elbs, Schenck, Smith, Wieland, and Story reactions is given. Unnamed rearrangements of organic peroxides and related processes are also analyzed. The rearrangements and related processes of important natural and synthetic peroxides are discussed separately. PMID:27559418

  2. 3D surface topology guides stem cell adhesion and differentiation.

    Science.gov (United States)

    Viswanathan, Priyalakshmi; Ondeck, Matthew G; Chirasatitsin, Somyot; Ngamkham, Kamolchanok; Reilly, Gwendolen C; Engler, Adam J; Battaglia, Giuseppe

    2015-06-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.

  3. Bioengineering approaches to guide stem cell-based organogenesis.

    Science.gov (United States)

    Gjorevski, Nikolche; Ranga, Adrian; Lutolf, Matthias P

    2014-05-01

    During organogenesis, various molecular and physical signals are orchestrated in space and time to sculpt multiple cell types into functional tissues and organs. The complex and dynamic nature of the process has hindered studies aimed at delineating morphogenetic mechanisms in vivo, particularly in mammals. Recent demonstrations of stem cell-driven tissue assembly in culture offer a powerful new tool for modeling and dissecting organogenesis. However, despite the highly organotypic nature of stem cell-derived tissues, substantial differences set them apart from their in vivo counterparts, probably owing to the altered microenvironment in which they reside and the lack of mesenchymal influences. Advances in the biomaterials and microtechnology fields have, for example, afforded a high degree of spatiotemporal control over the cellular microenvironment, making it possible to interrogate the effects of individual microenvironmental components in a modular fashion and rapidly identify organ-specific synthetic culture models. Hence, bioengineering approaches promise to bridge the gap between stem cell-driven tissue formation in culture and morphogenesis in vivo, offering mechanistic insight into organogenesis and unveiling powerful new models for drug discovery, as well as strategies for tissue regeneration in the clinic. We draw on several examples of stem cell-derived organoids to illustrate how bioengineering can contribute to tissue formation ex vivo. We also discuss the challenges that lie ahead and potential ways to overcome them.

  4. Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); S. Middendorp; K. Dahlenborg; A. Maas (Alex); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)

    2001-01-01

    textabstractBruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chai

  5. Bcl-1 gene rearrangements in mantle cell lymphoma : A comprehensive analysis of 118 cases, including B-5-fixed tissue, by polymerase chain reaction and southern transfer analysis

    NARCIS (Netherlands)

    Chibbar, R; Leung, K; McCormick, S; Ritzkalla, K; Strickler, J; Staggs, R; Poppema, S; Brunning, RD; McGlennen, RC

    1998-01-01

    We evaluated 118 cases of mantle cell lymphoma by polymerase chain reaction (PCR) for the major translocation cluster (MIG) region and another breakpoint corresponding to probe p94(PS), located 24 kb telomeric to the MTC locus on chromosome 11. The specimens included 64 frozen, 19 formalin-fixed, an

  6. A short guide to technology development in cell biology

    NARCIS (Netherlands)

    B. van Steensel (Bas)

    2015-01-01

    textabstractNew technologies drive progress in many research fields, including cell biology. Much of technological innovation comes from "bottom-up" efforts by individual students and postdocs. However, technology development can be challenging, and a successful outcome depends on many factors. This

  7. Guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata

    Directory of Open Access Journals (Sweden)

    Won-Gyu Bae

    2015-12-01

    Full Text Available Engineering complex extracellular matrix (ECM is an important challenge for cell and tissue engineering applications as well as for understanding fundamental cell biology. We developed the methodology for fabrication of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with original wrinkling technique for the generation of well-defined native ECM-like platforms by culturing fibroblast cells on the multiscale substrata [1]. This paper provides information on detailed characteristics of polyethylene glycol-diacrylate multiscale substrata. In addition, a possible model for guided extracellular matrix formation from fibroblast cells cultured on bio-inspired configurable multiscale substrata is proposed.

  8. Guiding plant virus particles to integrin-displaying cells

    Science.gov (United States)

    Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

    2012-05-01

    Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

  9. Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line.

    Science.gov (United States)

    Priesnitz, Christian; Niklas, Jens; Rose, Thomas; Sandig, Volker; Heinzle, Elmar

    2012-03-01

    This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.

  10. Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes.

    Science.gov (United States)

    Takata, T; Wang, H L; Miyauchi, M

    2001-10-01

    The purpose of this study was to evaluate the biological effects of guided tissue regeneration (GTR) membrane materials, per se, on the periodontal tissue regeneration. Rat periodontal ligament (PDL)-derived cells were used to study the attachment, proliferation and differentiation, in vitro, on various GTR membranes. Five commercially available membranes bovine type I collagen (BioMend; BM), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL) and expanded polytetrafluoroethylene: e-PTFE (Gore Tex; GT)-were examined. A 3 x 3 mm section of the membrane was fixed to the bottom of a 35 x 10 mm style culture dish and plated with 2 ml of cell suspension at an initial density of 5 x 10(4) cells/ml in culture medium with 10% fetal bovine serum. For cell growth analysis, the specimens were fixed with 10% buffered formalin and stained with hematoxylin at 1.5 hours and 1, 3 and 5 days after cell seeding. The number of cells included in a unit area of 0.25 mm2 were counted under light microscopy. As a comparative scaffold of cell proliferation, a plastic cover for cell culture slip (Celldesk; CD) was used. For analysis of cell differentiation, activity of alkaline phosphatase (ALP) and calcification were histochemically revealed after 2-week cultivation. The initial number of PDL cells attached to the membrane at 1.5 hours after cell seeding was different among membranes. RL, TG and EG had the same level of attached cell numbers as that on CD, while the cell numbers on GT and BM were significantly lower than that on CD (p membranes examined. RL and BM demonstrated a significantly higher number of cells at 5 days than at 1.5 hours (p 0.1). EG had a similar number of cell attachments to that at 1.5 hours throughout the experimental period. There was almost no cell proliferation on GT. Cell clusters of ALP positive cells and foci of calcification were seen on all membranes except for

  11. A short guide to technology development in cell biology.

    Science.gov (United States)

    van Steensel, Bas

    2015-03-16

    New technologies drive progress in many research fields, including cell biology. Much of technological innovation comes from "bottom-up" efforts by individual students and postdocs. However, technology development can be challenging, and a successful outcome depends on many factors. This article outlines some considerations that are important when embarking on a technology development project. Despite the challenges, developing a new technology can be extremely rewarding and could lead to a lasting impact in a given field.

  12. Stepwise-activable multifunctional peptide-guided prodrug micelles for cancerous cells intracellular drug release

    Science.gov (United States)

    Zhang, Jing; Li, Mengfei; Yuan, Zhefan; Wu, Dan; Chen, Jia-da; Feng, Jie

    2016-10-01

    A novel type of stepwise-activable multifunctional peptide-guided prodrug micelles (MPPM) was fabricated for cancerous cells intracellular drug release. Deca-lysine sequence (K10), a type of cell-penetrating peptide, was synthesized and terminated with azido-glycine. Then a new kind of molecule, alkyne modified doxorubicin (DOX) connecting through disulfide bond (DOX-SS-alkyne), was synthesized. After coupling via Cu-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction, reduction-sensitive peptide-guided prodrug was obtained. Due to the amphiphilic property of the prodrug, it can assemble to form micelles. To prevent the nanocarriers from unspecific cellular uptake, the prodrug micelles were subsequently modified with 2,3-dimethyl maleic anhydride to obtain MPPM with a negatively charged outer shell. In vitro studies showed that MPPM could be shielded from cells under psychological environment. However, when arriving at mild acidic tumor site, the cell-penetrating capacity of MPPM would be activated by charge reversal of the micelles via hydrolysis of acid-labile β-carboxylic amides and regeneration of K10, which enabled efficient internalization of MPPM by tumor cells as well as following glutathione- and protease-induced drug release inside the cancerous cells. Furthermore, since the guide peptide sequences can be accurately designed and synthesized, it can be easily changed for various functions, such as targeting peptide, apoptotic peptide, even aptamers, only need to be terminated with azido-glycine. This method can be used as a template for reduction-sensitive peptide-guided prodrug for cancer therapy.

  13. Shape and shear guide sperm cells spiraling upstream

    Science.gov (United States)

    Kantsler, Vasily; Dunkel, Jorn; Goldstein, Raymond E.

    2014-11-01

    A major puzzle in biology is how mammalian sperm determine and maintain the correct swimming direction during the various phases of the sexual reproduction process. Currently debated mechanisms for sperm long range travel vary from peristaltic pumping to temperature sensing (thermotaxis) and direct response to fluid flow (rheotaxis), but little is known quantitatively about their relative importance. Here, we report the first quantitative experimental study of mammalian sperm rheotaxis. Using microfluidic devices, we investigate systematically the swimming behavior of human and bull sperm over a wide range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions and chirality of the flagellar beat leads to a stable upstream spiraling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilization. To rationalize these findings, we identify a minimal mathematical model that is capable of describing quantitatively the experimental observations.

  14. Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

    Science.gov (United States)

    Unachukwu, Uchenna John; Warren, Alice; Li, Ze; Mishra, Shawn; Zhou, Jing; Sauane, Moira; Lim, Hyungsik; Vazquez, Maribel; Redenti, Stephen

    2016-03-01

    To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α - CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

  15. Complex chromosomal rearrangements by single catastrophic pathogenesis in NUT midline carcinoma

    Science.gov (United States)

    Lee, J.-K.; Louzada, S.; An, Y.; Kim, S. Y.; Kim, S.; Youk, J.; Park, S.; Koo, S. H.; Keam, B.; Jeon, Y. K.; Ku, J.-L.; Yang, F.; Kim, T. M.

    2017-01-01

    Background Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare aggressive malignancy often occurring in the tissues of midline anatomical structures. Except for the pathognomonic BRD3/4–NUT rearrangement, the comprehensive landscape of genomic alterations in NMCs has been unexplored. Patients and methods We investigated three NMC cases, including two newly diagnosed NMC patients in Seoul National University Hospital, and a previously reported cell line (Ty-82). Whole-genome and transcriptome sequencing were carried out for these cases, and findings were validated by multiplex fluorescence in situ hybridization and using individual fluorescence probes. Results Here, we present the first integrative analysis of whole-genome sequencing, transcriptome sequencing and cytogenetic characterization of NUT midline carcinomas. By whole-genome sequencing, we identified a remarkably similar pattern of highly complex genomic rearrangements (previously denominated as chromoplexy) involving the BRD3/4–NUT oncogenic rearrangements in two newly diagnosed NMC cases. Transcriptome sequencing revealed that these complex rearrangements were transcribed as very simple BRD3/4–NUT fusion transcripts. In Ty-82 cells, we also identified a complex genomic rearrangement involving the BRD4–NUT rearrangement underlying the simple t(15;19) karyotype. Careful inspections of rearrangement breakpoints indicated that these rearrangements were likely attributable to single catastrophic events. Although the NMC genomes had >3000 somatic point mutations, canonical oncogenes or tumor suppressor genes were rarely affected, indicating that they were largely passenger events. Mutational signature analysis showed predominant molecular clock-like signatures in all three cases (accounting for 54%−75% of all base substitutions), suggesting that NMCs may arise from actively proliferating normal cells. Conclusion Taken together, our findings suggest that a single catastrophic event in

  16. Chromosome 12;15 rearrangements in patients with recurrent miscarriage

    Directory of Open Access Journals (Sweden)

    Nair S

    2006-01-01

    Full Text Available Background: An abnormal karyotype in either partner, especially featuring a translocation and/or inversion is considered to be a cause of recurrent miscarriages. It is generally assumed that recurrent miscarriage might be due to recurrent chromosomal abnormalities in the fetus due to a balanced aberration in one of the parents being inherited by the offspring in an unbalanced form. Aim: Evaluation of chromosomal rearrangements in couples with recurrent miscarriages. Materials and Methods: Peripheral blood was collected and lymphocyte cultures were set up. Slides prepared from the cell suspension were stained and screened for metaphases followed by karyotyping. Result: Balanced translocation was observed in the male partner in one case and in the female partners in the three other cases. Conclusion: Couples with recurrent miscarriage should be investigated for chromosomal rearrangements, thus helping in genetic counseling and providing the options for future pregnancies.

  17. RNA-Guided DNA Rearrangements in Breast Cancer

    Science.gov (United States)

    2012-09-01

    in the developing macronucleus. Immunofluorescence suggests that Otiwi1 is absent from early conjugating pairs, when micronuclei begin meiosis ...Lepere, G., Nowacki, M., Serrano, V., Gout, J.F., Guglielmi, G., Duharcourt, S., and Meyer, E. (2009). Silencing-associated and meiosis -specific...macronucleus to the developing macronucleus. Otiwi1 is absent at the initial pairing stage when the micronucleus (MIC) begins meiosis (A), but accumulates in

  18. 非小细胞肺癌EML4-ALK融合基因检测技术研究进展%Advances in assay of EML4-ALK gene rearrangements in non-small-cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    陈健; 李治桦; 刘晓晴

    2014-01-01

    Echinoderm microtubule associated protein like 4-anaplastic lymphomakinase ( EML4-ALK) rearrangement is another driver-mutation in non-small-cell lung cancer (NSCLC) besides epidermal growth factor receptor (EGFR).Accu-rate identification of EML 4-ALK lung adenocarcinoma is essential for the selection of appropriate therapy .Immunohisto-chemistry (IHC), fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR) are the main three assay methods for clinical practice .Recently, researchers have done lots of work on the exploration of detection , diagnosis, and treatment prediction of carcinoma based on serum proteomics .In this paper, recent developments in these fields are reviewed.%棘皮动物微管相关蛋白4-间变性淋巴瘤激酶( echinoderm microtubule associated protein like 4-anaplastic lymphomakinase, EML4-ALK)融合基因是继表皮生长因子受体(epidermal growth factor receptor ,EGFR)之后的又一非小细胞肺癌驱动基因。 EML4-ALK基因检测对临床治疗至关重要。目前,EML4-ALK基因的检测方法包括免疫组化、免疫荧光原位杂交、基于PCR扩增方法等。近年来,国内外学者对血清蛋白质组学在肿瘤检测、诊断、疗效预测方面做了大量有益的探索。该文综述了EML4-ALK的检测方法以及ALK血清蛋白质组学等方面的研究进展。

  19. Transesophageal echocardiography-guided thrombectomy of intracardiac renal cell carcinoma without cardiopulmonary bypass

    Science.gov (United States)

    Souki, Fouad Ghazi; Demos, Michael; Fermin, Lilibeth; Ciancio, Gaetano

    2016-01-01

    Advanced renal cell carcinoma (RCC) resection has important anesthetic management implications, particularly when tumor extends, suprahepatic, into the right atrium. Use of transesophageal echocardiogram (TEE) is essential in identifying tumor extension and guiding resection. Latest surgical approach avoids venovenous and cardiopulmonary bypass yet requires special precautions and interventions on the anesthesiologist's part. We present a case of Level IV RCC resected without cardiopulmonary bypass and salvaged by TEE guidance and detection of residual intracardiac tumor. PMID:27716710

  20. Quantifying stretching and rearrangement in epithelial sheet migration

    CERN Document Server

    Lee, Rachel M; Nordstrom, Kerstin N; Ouellette, Nicholas T; Losert, Wolfgang; 10.1088/1367-2630/15/2/025036

    2013-01-01

    Although understanding the collective migration of cells, such as that seen in epithelial sheets, is essential for understanding diseases such as metastatic cancer, this motion is not yet as well characterized as individual cell migration. Here we adapt quantitative metrics used to characterize the flow and deformation of soft matter to contrast different types of motion within a migrating sheet of cells. Using a Finite-Time Lyapunov Exponent (FTLE) analysis, we find that - in spite of large fluctuations - the flow field of an epithelial cell sheet is not chaotic. Stretching of a sheet of cells (i.e., positive FTLE) is localized at the leading edge of migration. By decomposing the motion of the cells into affine and non-affine components using the metric D$^{2}_{min}$, we quantify local plastic rearrangements and describe the motion of a group of cells in a novel way. We find an increase in plastic rearrangements with increasing cell densities, whereas inanimate systems tend to exhibit less non-affine rearran...

  1. Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves.

    Science.gov (United States)

    Cattin, Anne-Laure; Burden, Jemima J; Van Emmenis, Lucie; Mackenzie, Francesca E; Hoving, Julian J A; Garcia Calavia, Noelia; Guo, Yanping; McLaughlin, Maeve; Rosenberg, Laura H; Quereda, Victor; Jamecna, Denisa; Napoli, Ilaria; Parrinello, Simona; Enver, Tariq; Ruhrberg, Christiana; Lloyd, Alison C

    2015-08-27

    The peripheral nervous system has remarkable regenerative capacities in that it can repair a fully cut nerve. This requires Schwann cells to migrate collectively to guide regrowing axons across a 'bridge' of new tissue, which forms to reconnect a severed nerve. Here we show that blood vessels direct the migrating cords of Schwann cells. This multicellular process is initiated by hypoxia, selectively sensed by macrophages within the bridge, which via VEGF-A secretion induce a polarized vasculature that relieves the hypoxia. Schwann cells then use the blood vessels as "tracks" to cross the bridge taking regrowing axons with them. Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair. This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.

  2. Langerhans Cell Histiocytosis Arising from the Mandible as Diagnosed by US-guided Core Biopsy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Soo Jin [Center of Thyroid Cancer, National Cancer Center, Goyang (Korea, Republic of); Kim, Eun Kyung [Research Institute of Radiological Science, Yonsei University Heath System, Seoul (Korea, Republic of); Lee, Min Kyung [Eulji University College of Medicine, Eulji University Hospital, Daejeon (Korea, Republic of)

    2010-09-15

    Langerhans cell histiocytosis (LCH) is a clonal proliferative disorder of Langerhans cells. Although LCH is not considered a malignant disease, its appearance on radiographs may be similar to that of a malignant tumor. The diagnosis of LCH is usually made by a soft tissue biopsy, or by bone marrow aspiration or curettage. We present a patient with a mandibular mass confirmed to be LCH by US-guided core needle biopsy, and present a strategy for diagnosing localized LCH of the bone based on the usefulness and reliability of the percutaneous biopsy

  3. A photolabile hydrogel for guided three-dimensional cell growth and migration

    Science.gov (United States)

    Luo, Ying; Shoichet, Molly S.

    2004-04-01

    Tissue engineering aims to replace, repair or regenerate tissue/organ function, by delivering signalling molecules and cells on a three-dimensional (3D) biomaterials scaffold that supports cell infiltration and tissue organization. To control cell behaviour and ultimately induce structural and functional tissue formation on surfaces, planar substrates have been patterned with adhesion signals that mimic the spatial cues to guide cell attachment and function. The objective of this study is to create biochemical channels in 3D hydrogel matrices for guided axonal growth. An agarose hydrogel modified with a cysteine compound containing a sulphydryl protecting group provides a photolabile substrate that can be patterned with biochemical cues. In this transparent hydrogel we immobilized the adhesive fibronectin peptide fragment, glycine-arginine-glycine-aspartic acid-serine (GRGDS), in selected volumes of the matrix using a focused laser. We verified in vitro the guidance effects of GRGDS oligopeptide-modified channels on the 3D cell migration and neurite outgrowth. This method for immobilizing biomolecules in 3D matrices can generally be applied to any optically clear hydrogel, offering a solution to construct scaffolds with programmed spatial features for tissue engineering applications.

  4. Ultrasound guided electrochemotherapy for the treatment of a clear cell thymoma in a cat

    Science.gov (United States)

    Spugnini, Enrico Pierluigi; Menicagli, Francesco; Pettorali, Michela; Baldi, Alfonso

    2017-01-01

    A twelve-year-old male castrated domestic shorthair cat was presented for rapidly progressing respiratory distress. The cat was depressed, tachypneic and moderately responsive. Ultrasonography showed a mediastinal mass associated with a significant pleural effusion that needed tapping every five to seven days. Ultrasound guided biopsy yielded a diagnosis of clear cell thymoma upon histopathology. After complete staging procedures, the owner elected to treat the cat with electrochemotherapy (ECT) using systemic bleomycin. Two sessions of ultrasound guided ECT were performed at two week intervals with trains of biphasic electric pulses applied using needle electrodes until complete coverage of the area was achieved. The treatment was well tolerated and resulted in partial remission (PR). Additional sessions were performed on a monthly basis. The cat is still in PR after fourteen months. ECT resulted in improved local control and should be considered among the available adjuvant treatments in pets carrying visceral tumors. PMID:28331834

  5. Tracking epithelial cell junctions in C. elegans embryogenesis with active contours guided by SIFT flow.

    Science.gov (United States)

    Kang, Sukryool; Lee, Chen-Yu; Gonçalves, Monira; Chisholm, Andrew D; Cosman, Pamela C

    2015-04-01

    Quantitative analysis of cell shape in live samples is an important goal in developmental biology. Automated or semi-automated segmentation and tracking of cell nuclei has been successfully implemented in several biological systems. Segmentation and tracking of cell surfaces has been more challenging. Here, we present a new approach to tracking cell junctions in the developing epidermis of C. elegans embryos. Epithelial junctions as visualized with DLG-1::GFP form lines at the subapical circumference of differentiated epidermal cells and delineate changes in epidermal cell shape and position. We develop and compare two approaches for junction segmentation. For the first method (projection approach), 3-D cell boundaries are projected into 2D for segmentation using active contours with a nonintersecting force, and subsequently tracked using scale-invariant feature transform (SIFT) flow. The resulting 2-D tracked boundaries are then back-projected into 3-D space. The second method (volumetric approach) uses a 3-D extended version of active contours guided by SIFT flow in 3-D space. In both methods, cell junctions are manually located at the first time point and tracked in a fully automated way for the remainder of the video. Using these methods, we have generated the first quantitative description of ventral epidermal cell movements and shape changes during epidermal enclosure.

  6. BIOMED-2系统引物用于检测T细胞淋巴瘤石蜡样本T细胞受体γ基因重排的研究%Application of BIOMED-2 primers in analysis of T-cell receptor γ gene rearrangements in paraffin-embedded tissue specimens of T-cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    唐源; 江炜; 李雷; 纪洪; 李韵; 李甘地; 刘卫平

    2009-01-01

    目的 了解BIOMED-2系统T细胞受体(TCR)γ引物组合对T细胞淋巴瘤的常规石蜡包埋组织样本中TCR基因重排的检出情况及其实用性.方法 用酚/氯仿法提取55例各种组织类型的T细胞淋巴瘤石蜡包埋组织样本的DNA并通过扩增看家基因β-globin检测其质量,利用BIOMED-2系统TCR-γ引物组合和TCR-γ基因通用型引物(TVG/TJX)对55例进行TCR基因重排检测,比较二者的检出率并进行统计学分析.结果 BIOMED-2系统TCR-γ引物组合和TCRγ基因通用型引物(TVG/TJX)的TCR基因重排检出率分别为76.4%和60.0%,前者高于后者,二者的差异无统计学意义(P>0.05).结论 BIOMED-2系统TCRγ引物组合适用于本组T细胞淋巴瘤石蜡包埋组织样本的TCR基因重排检测.%Objective To evaluate the practical values of PCR detectable T-cell receptor (TCR) gene rearrangement in paraffin embedded tissue samples in the diagnosis of T-cell malignancies using BIOMED-2 PCR multiplex tubes TCRγ(A + B). Methods Traditional phenol-chloroform method was used to extract DNA from 55 cases of archival paraffin embedded tissues samples of T-cell malignancies and the DNA quality was evaluated by PCR-based amplification of housekeeping gone β-globin. The selected BIOMED-2 PCR multiplex tubes TCRγ(A + B) were used to detect TCR gene rearrangement and comparison with the results of universal TCR primers (TVG/TJX) was performed. Results Positive detection rates by the BIOMED-2 multiplex tubes TCRγ(A + B) and the universal primers (TVG/TJX) were 76.4% and 60.0%, respectively. There were not statistical difference between the methods (P > 0.05). Conclusion BIOMED-2 multiplex tubes TCRγ(A + B) is suitable for detection of clonal rearrangements of TCR genes in current archival paraffin embeded tissue samples of T-cell malignancies.

  7. Chromosomal rearrangements and the pathogenesis of differentiated thyroid cancer

    Directory of Open Access Journals (Sweden)

    Stefan K.G. Grebe

    2011-12-01

    Full Text Available The majority of thyroid cancers arise from the follicular cells of the thyroid gland, which yield a wide variety of distinct morphotypes, ranging from relatively indolent lesions to the most malignant forms of cancer known. The remaining primary thyroid cancers arise from C cells within the gland and result primarily from mutations of the RET protooncogene, germ line mutations of which give rise to the various forms of multiple endocrine neoplasia. The most common of the follicular cell-derived cancers are papillary carcinomas, (PTC, followed by follicular carcinomas (FTC and its Hurthle cell variant (HCC and finally anaplastic carcinomas (ATC. The pathogenesis of many thyroid cancers, of both PTC and FTC morphotype, involves chromosomal translocations. Rearrangements of the RET protoconcogene are known to be involved in the pathogenesis of ca. 50% of PTC. A similar proportion of FTC have been associated with a t(2;3(q13;p25 translocation, fusing the thyroid-specific transcription factor PAX8 with the peroxisome proliferator-activated receptor gamma (PPARγ nuclear receptor, a ubiquitously expressed transcription factor. These rearrangements have analogy with translocations in erythropoetic cells, which form the only other known group of human malignancies that are largely the result of chromosomal translocation events. In this review we compare and contrast the oncogenic properties of thyroid and erythroid chromosomal transformations and speculate on mechanisms leading to their formation.

  8. Sex chromosome rearrangements in Polyphaga beetles.

    Science.gov (United States)

    Dutrillaux, A M; Dutrillaux, B

    2009-01-01

    The presence of a parachute sex chromosome bivalent (Xyp) at metaphase I of male meiosis is a well-known characteristic of Coleoptera, present in almost all families of this order and assumed to represent their ancestral sex chromosome formula. Sex chromosomes appear to be manifold more frequently involved in inter-chromosomal rearrangements than the average of the nine autosomal pairs usually forming their karyotype. This leads to various formulae such as neo-sex, multiple sex and perhaps unique sex chromosomes. These rearrangements alter the intimate association between sex chromosomes and nucleolar proteins, which are usual components of the Xyp. Different situations, selected in a series of 125 mitotic and meiotic cytogenetic studies of Polyphaga beetle species, are reported and discussed, with the aim to improve our knowledge on the mechanisms of sex chromosome rearrangements, the relationships with nucleoli and the consequences on dosage compensation and chromosome segregation.

  9. Genomic regulatory landscapes and chromosomal rearrangements

    DEFF Research Database (Denmark)

    Ladegaard, Elisabete L Engenheiro

    2008-01-01

    The main objectives of the PhD study are to identify and characterise chromosomal rearrangements within evolutionarily conserved regulatory landscapes around genes involved in the regulation of transcription and/or development (trans-dev genes). A frequent feature of trans-dev genes...... the complex spatio-temporal expression of the associated trans-dev gene. Rare chromosomal breakpoints that disrupt the integrity of these regulatory landscapes may be used as a tool, not only to make genotype-phenotype associations, but also to link the associated phenotype with the position and tissue...... specificity of the individual CNEs. In this PhD study I have studied several chromosomal rearrangements with breakpoints in the vicinity of trans-dev genes. This included chromosomal rearrangements compatible with known phenotype-genotype associations (Rieger syndrome-PITX2, Mowat-Wilson syndrome-ZEB2...

  10. Assessment of T-cell clonality via T-cell receptor-gamma rearrangements in cutaneous T-cell-dominant infiltrates using polymerase chain reaction and single-stranded DNA conformational polymorphism assay.

    Science.gov (United States)

    Chen, Michael; Deng, April; Crowson, A Neil; Srinivasan, Mythily; Yearsley, Kurtis H; Jewell, Scott; Morrison, Carl; Long, Susan; Werling, Robert; Magro, Cynthia

    2004-12-01

    Discerning the pathologic significance of cutaneous T-cell infiltrates can pose a diagnostic challenge for dermatopathologists. Reactive conditions such as drug-associated lymphomatoid hypersensitivity and lymphomatoid lupus erythematosus can demonstrate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pigmentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent malignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are reports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single-stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell-rich biopsies to determine the relative specificity and sensitivity of this methodology. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed without LCM. We found that monoclonality or oligoclonality is 86% specific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular profile mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP.

  11. Guiding osteogenesis of mesenchymal stem cells using carbon-based nanomaterials

    Science.gov (United States)

    Kang, Ee-Seul; Kim, Da-Seul; Suhito, Intan Rosalina; Choo, Sung-Sik; Kim, Seung-Jae; Song, Inbeom; Kim, Tae-Hyung

    2017-01-01

    In the field of regenerative medicine, stem cells are highly promising due to their innate ability to generate multiple types of cells that could replace/repair damaged parts of human organs and tissues. It has been reported that both in vitro and in vivo function/survival of stem cells could significantly be improved by utilizing functional materials such as biodegradable polymers, metal composites, nanopatterns and nanohybrid particles. Of various biocompatible materials available for use in stem cell-based therapy and research, carbon-based materials—including fullerenes graphene/graphene oxide and carbon nanotubes—have been found to possess unique physicochemical characteristics that contribute to the effective guidance of stem cell differentiation into specific lineages. In this review, we discuss a number of previous reports that investigated the use of carbon-based materials to control stem cell behavior, with a particular focus on their immense potential to guide the osteogenesis of mesenchymal stem cells (MSCs). We hope that this review will provide information on the full potential of using various carbon-based materials in stem cell-mediated regenerative therapy, particularly for bone regeneration and repair.

  12. Light-Addressable Electrodeposition of Magnetically-Guided Cells Encapsulated in Alginate Hydrogels for Three-Dimensional Cell Patterning

    Directory of Open Access Journals (Sweden)

    Shih-Hao Huang

    2014-11-01

    Full Text Available This paper describes a light-addressable electrolytic system used to perform an electrodeposition of magnetically-guided cells encapsulated in alginate hydrogels using a digital micromirror device (DMD for three-dimensional cell patterning. In this system, the magnetically-labeled cells were first manipulated into a specific arrangement by changing the orientation of the magnetic field, and then a patterned light illumination was projected onto a photoconductive substrate serving as a photo-anode to cause gelation of calcium alginate through sol-gel transition. By controlling the illumination pattern on the DMD, we first successfully produced cell-encapsulated multilayer alginate hydrogels with different shapes and sizes in each layer via performing multiplexed micropatterning. By combining the magnetically-labeled cells, light-addressable electrodeposition, and orientation of the magnetic fields, we have successfully demonstrated to fabricate two layers of the cell-encapsulated alginate hydrogels, where cells in each layer can be manipulated into cross-directional arrangements that mimic natural tissue. Our proposed method provides a programmable method for the spatiotemporally controllable assembly of cell populations into three-dimensional cell patterning and could have a wide range of biological applications in tissue engineering, toxicology, and drug discovery.

  13. Rearrangement of cluster structure during fission processes

    DEFF Research Database (Denmark)

    Lyalin, Andrey G.; Obolensky, Oleg I.; Solov'yov, Andrey V.

    2004-01-01

    Results of molecular dynamics simulations of fission reactions $Na_10^2+ -->Na_7^++ Na_3^+ and Na_18^2+--> 2Na_9^+ are presented. The dependence of the fission barriers on the isomer structure of the parent cluster is analysed. It is demonstrated that the energy necessary for removing homothetic...... groups of atoms from the parent cluster is largely independent of the isomer form of the parent cluster. The importance of rearrangement of the cluster structure during the fission process is elucidated. This rearrangement may include transition to another isomer state of the parent cluster before actual...

  14. Image-guided precision manipulation of cells and nanoparticles in microfluidics

    Science.gov (United States)

    Cummins, Zachary

    Manipulation of single cells and particles is important to biology and nanotechnology. Our electrokinetic (EK) tweezers manipulate objects in simple microfluidic devices using gentle fluid and electric forces under vision-based feedback control. In this dissertation, I detail a user-friendly implementation of EK tweezers that allows users to select, position, and assemble cells and nanoparticles. This EK system was used to measure attachment forces between living breast cancer cells, trap single quantum dots with 45 nm accuracy, build nanophotonic circuits, and scan optical properties of nanowires. With a novel multi-layer microfluidic device, EK was also used to guide single microspheres along complex 3D trajectories. The schemes, software, and methods developed here can be used in many settings to precisely manipulate most visible objects, assemble objects into useful structures, and improve the function of lab-on-a-chip microfluidic systems.

  15. Guiding intracortical brain tumour cells to an extracortical cytotoxic hydrogel using aligned polymeric nanofibres

    Science.gov (United States)

    Jain, Anjana; Betancur, Martha; Patel, Gaurangkumar D.; Valmikinathan, Chandra M.; Mukhatyar, Vivek J.; Vakharia, Ajit; Pai, S. Balakrishna; Brahma, Barunashish; MacDonald, Tobey J.; Bellamkonda, Ravi V.

    2014-03-01

    Glioblastoma multiforme is an aggressive, invasive brain tumour with a poor survival rate. Available treatments are ineffective and some tumours remain inoperable because of their size or location. The tumours are known to invade and migrate along white matter tracts and blood vessels. Here, we exploit this characteristic of glioblastoma multiforme by engineering aligned polycaprolactone (PCL)-based nanofibres for tumour cells to invade and, hence, guide cells away from the primary tumour site to an extracortical location. This extracortial sink is a cyclopamine drug-conjugated, collagen-based hydrogel. When aligned PCL-nanofibre films in a PCL/polyurethane carrier conduit were inserted in the vicinity of an intracortical human U87MG glioblastoma xenograft, a significant number of human glioblastoma cells migrated along the aligned nanofibre films and underwent apoptosis in the extracortical hydrogel. Tumour volume in the brain was significantly lower following insertion of aligned nanofibre implants compared with the application of smooth fibres or no implants.

  16. Limited junctional diversity of V delta 5-J delta 1 rearrangement in multiple sclerosis patients.

    Science.gov (United States)

    Nowak, J S; Michałowska-Wender, G; Januszkiewicz, D; Wender, M

    1997-01-01

    T-cell receptor (TCR) delta gene repertoire, as assessed by V delta-J delta rearrangements, has been analyzed in nine multiple sclerosis (MS) cases and in 30 healthy individuals by seminested PCR technique. Among the V delta-J delta junctional diversities studied, the most striking result has been observed in V delta 5-J delta 1 rearrangement. The detection of repeated V delta 5-J delta 1 nucleotide sequences in all analyzed clones from seven out of nine patients studied proved the monoclonal nature of gamma delta T-cells with V delta 5-J delta 1 rearrangement. The clonal nature of this rearrangement proved by PAGE and sequencing analysis may suggest an antigen-driven expansion of gamma delta T cells and argues for a significant role of gamma delta T-cells with V delta 5-J delta 1 rearrangement in MS pathogenesis. However, it cannot be excluded that clonal expansion of these lymphocytes may represent secondary change to central nervous system damage.

  17. Complications of ultrasound-guided percutaneous microwave ablation of renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Dong XJ

    2016-09-01

    Full Text Available Xuejuan Dong, Xin Li, Jie Yu, Ming-an Yu, Xiaoling Yu, Ping Liang Department of Interventional Ultrasound, Chinese PLA General Hospital, Beijing, People’s Republic of China Purpose: To retrospectively review the complications of ultrasound (US-guided percutaneous microwave ablation (MWA of renal cell carcinoma.Patients and methods: In this study, 101 patients with 105 tumors seen from April 2006 to Feb 2014 were enrolled retrospectively. The patients were treated with US-guided percutaneous MWA and were followed up with contrast-enhanced US and computed tomography or magnetic resonance imaging at 1, 3, and 6 months and every 6 months thereafter.Results: Technical success was achieved in 99 of 105 tumors (94.3%. The median follow-up time was 25 (range 1.13–93.23 months. Among the 105 tumors, 26 complications in 24.8% of patients and 23 minor complications (Clavien–Dindo Grades I and II in 21.9% of patients were noted, accounting for 88.5% of all complications. All the minor complications were cured. Three major complications (Clavien–Dindo Grade ≥III occurred in 2.9% of the patients, accounting for 11.5% of all complications: hydrothorax in two patients and bowel injury in one. The two patients who had hydrothorax post-MWA had a history of cirrhosis and were treated with catheter drainage. The bowel injury was treated surgically. In all patients, the changes in serum creatinine and urea nitrogen levels from before to after the procedure were small.Conclusion: US-guided percutaneous MWA is a beneficial treatment for renal cell carcinoma in selected patients; however, if the renal tumor is close to the bowel, or the patient has serious comorbidities or has undergone abdominal surgery, the procedure must be performed more carefully. Keywords: microwave ablation, renal cell, complications, carcinoma

  18. Cytometric measurement of cell proliferation in echo-guided biopsies from focal lesions of the liver.

    Science.gov (United States)

    Faccioli, S; Chieco, P; Gramantieri, L; Stecca, B A; Bolondi, L

    1996-02-01

    Increased proliferative activity determined in surgical specimens of hepatocellular carcinoma (HCC) has been associated with tumor grade and patient survival. The measurement of cell proliferation in echo-guided biopsies of small focal liver lesions might provide useful information for the early recognition of malignancy and for predicting the aggressiveness of small HCCs. We assessed the diagnostic and prognostic value of cell proliferation in 91 echo-guided needle biopsies of focal liver lesions using the monoclonal antibody Ki-67, which detects a human nuclear antigen that is present in proliferating cells. Measurements were performed by image cytometry as the percentage of Ki-67 positive hepatocytes nuclei over total hepatocyte nuclei in the biopsy. At the histological examination, 27 lesions were diagnosed as chronic hepatitis, 10 as cirrhosis, 11 as macroregenerative nodule, and 43 as HCC in cirrhotic liver. Although the highest Ki-67 values (> 20%) were found in less-differentiated HCCs, most well-differentiated HCCs and nine borderline nodules were completely devoid of Ki-67-positive hepatocytes. A sustained Ki-67 labeling (up to 16%) was found in hepatitis and cirrhosis, similar to that found in several malignant tumors. In the HCC subset, Ki-67 labeling was strongly correlated to the Edmondson-Steiner histological grade. However, survival analysis did not indicate a better outcome for those patients with low-proliferating tumors.

  19. Thermometry of Guided Molecular Beams from a Cryogenic Buffer-Gas Cell

    CERN Document Server

    Wu, X; Zeppenfeld, M; Chervenkov, S; Rempe, G

    2016-01-01

    We present a comprehensive characterization of cold molecular beams from a cryogenic buffer-gas cell, providing an insight into the physics of buffer-gas cooling. Cold molecular beams are extracted from a cryogenic cell by electrostatic guiding, which is also used to measure their velocity distribution. Molecules' rotational-state distribution is probed via radio-frequency resonant depletion spectroscopy. With the help of complete trajectory simulations, yielding the guiding efficiency for all of the thermally populated states, we are able to determine both the rotational and the translational temperature of the molecules at the output of the buffer-gas cell. This thermometry method is demonstrated for various regimes of buffer-gas cooling and beam formation as well as for molecular species of different sizes, $\\rm{CH_3F}$ and $\\rm{CF_3CCH}$. Comparison between the rotational and translational temperatures provides evidence of faster rotational thermalization for the $\\rm{CH_3F-He}$ system in the limit of low...

  20. Langerhans` cell histiocytosis of the spine: use of MRI in guiding biopsy

    Energy Technology Data Exchange (ETDEWEB)

    Kaplan, G.R.; Saifuddin, A. [Department of Diagnostic Imaging, Royal National Orthopaedic Hospital Trust, Brockley Hill (United Kingdom); Pringle, J.A.S. [Department of Morbid Anatomy, Royal National Orthopaedic Hospital Trust, Brockley Hill (United Kingdom); Noordeen, M.H.; Mehta, M.H. [Department of Spinal Deformities, Royal National Orthopaedic Hospital Trust, Brockley Hill (United Kingdom)

    1998-12-01

    The MRI features of two cases of spinal Langerhans` cell histiocytosis with multilevel involvement are presented in which MRI was of help in differentiating active from inactive healing lesions by the demonstration of signal changes in the vertebral body marrow of the active lesion, manifest as low signal intensity on T1-weighted sequences and high signal intensity on T2-weighted sequences. This distinction could not be made by plain radiography or bone scintigraphy. In cases where biopsy is required for diagnosis, MRI is recommended to guide the biopsy towards levels suggestive of active involvement. (orig.) With 7 figs., 13 refs.

  1. MRI-guided percutaneous laser ablation of small renal cell carcinoma: Initial clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Kariniemi, Juho; Ojala, Risto; Hellstroem, Pekka; Sequeiros, Roberto Blanco (Dept. of Radiology, Dept. of Surgery, Oulu Univ. Hospital, Oulu (Finland)), e-mail: juho.kariniemi@oulu.fi

    2010-05-15

    Background: The number of detected small renal cell carcinomas (RCCs) has been rising, largely due to advances in imaging. Open surgical resection is the standard management of small RCCs; however, imaging-guided percutaneous ablative therapies have emerged as a minimally invasive treatment alternative, especially for patients who are poor candidates for surgery. Purpose: To evaluate the initial clinical experience of magnetic resonance imaging (MRI)-guided percutaneous laser ablation of small RCCs. Material and Methods: Eight patients with 10 tumors were treated with percutaneous MRI-guided laser ablation. All tumors (diameter range 1.5-3.8 cm, mean 2.7 cm) were biopsy-proven RCCs. By using a 0.23 T open MRI system and general anesthesia in patients, one to four (mean 2.6) laser fibers were placed and the tumors were ablated under near real-time MRI control by observing the signal void caused by the temperature change in the heated tissue. The treatment was considered successful if the tumor showed no contrast enhancement at follow-up imaging. Results: All except one tumor were successfully ablated in one session. The first patient treated showed enhancing residual tumor in post-procedural MRI; she has thus far declined retreatment. One complication, a myocardial infarction, occurred; all other patients tolerated the procedure well. No local recurrence was discovered during the follow-up (range 12-30 months, mean 20 months). Conclusion: In this small group of patients with relatively short follow-up period, MRI-guided percutaneous laser ablation proved to be a promising treatment option for small RCCs

  2. Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer.

    Science.gov (United States)

    Robinson, Dan R; Kalyana-Sundaram, Shanker; Wu, Yi-Mi; Shankar, Sunita; Cao, Xuhong; Ateeq, Bushra; Asangani, Irfan A; Iyer, Matthew; Maher, Christopher A; Grasso, Catherine S; Lonigro, Robert J; Quist, Michael; Siddiqui, Javed; Mehra, Rohit; Jing, Xiaojun; Giordano, Thomas J; Sabel, Michael S; Kleer, Celina G; Palanisamy, Nallasivam; Natrajan, Rachael; Lambros, Maryou B; Reis-Filho, Jorge S; Kumar-Sinha, Chandan; Chinnaiyan, Arul M

    2011-11-20

    Breast cancer is a heterogeneous disease that has a wide range of molecular aberrations and clinical outcomes. Here we used paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers have a variety of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving genes encoding microtubule-associated serine-threonine kinase (MAST) and members of the Notch family. Both MAST and Notch-family gene fusions have substantial phenotypic effects in breast epithelial cells. Breast cancer cell lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and overexpression of MAST1 or MAST2 gene fusions has a proliferative effect both in vitro and in vivo. These findings show that recurrent gene rearrangements have key roles in subsets of carcinomas and suggest that transcriptome sequencing could identify individuals with rare, targetable gene fusions.

  3. Molecular Insights in MLL Rearranged Acute Leukemia

    NARCIS (Netherlands)

    R.W. Stam (Ronald)

    2006-01-01

    textabstractAcute lymphoblastic leukemia (ALL) in infants (<1 year of age) is characterized by a high incidence (~80%) of rearrangements of the MLL gene, resistance to several important chemotherapeutic drugs, and a poor treatment outcome. With overall survival rates for infant ALL not exceeding 50%

  4. High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells

    Science.gov (United States)

    Bian, Shengtai; Zhou, Yicen; Hu, Yawei; Cheng, Jing; Chen, Xiaofang; Xu, Youchun; Liu, Peng

    2017-01-01

    Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells. Two plasmids encoding enhanced green and red fluorescent proteins (EGFP and ERFP), respectively, were successfully electroporated into attached HeLa cells on a 169-microwell array chip with transfection efficiencies of 71.6 ± 11.4% and 62.9 ± 2.7%, and a cell viability above 95%. We also successfully conducted selective electroporation of sgRNA into 293T cells expressing the Cas9 nuclease in a high-throughput manner and observed the four-fold increase of the GFP intensities due to the repair of the protein coding sequences mediated by the CRISPR/Cas9 system. This study proved that this HiCEP system has the great potential to be used for arrayed functional screens with genome-wide CRISPR libraries on hard-to-transfect cells in the future. PMID:28211892

  5. Cellular reactions of osteoblast-like cells to a novel nanocomposite membrane for guided bone regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Meng Yao [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Department of Orthodontics, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Liu Man [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Stomatology Health Care Center, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen 518048 (China); Wang Shaoan [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Mo Anchun [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China)], E-mail: moanchun@163.com; Huang, Cui [State Key Laboratory of Oral Diseases, West China Stomatology Hospital, Sichuan University, Chengdu 610041 (China); Zuo Yi; Li Jidong [Research Center for Nano-biomaterials, Sichuan University, Chengdu 610041 (China)

    2008-11-15

    This study investigated the bioactivity and biocompatibility of hydroxyapatite nanoparticles (n-HA)/Polyamide-66 (PA66) nanocomposite membrane and expanded-polytetrafluoroethylene (e-PTFE) membrane (as control) to MG63 osteoblast-like cells. The attachment and proliferation of the cells on the porous surface of nHA/PA66 membrane and the surface of e-PTFE membrane were evaluated by scanning electron microscope (SEM) observation and the MTT assay. The bioactivity of the cells on the surface of the two membranes was evaluated by testing cell viability and alkaline phosphatase (ALP) activities. The results suggested that the bioresponse of MG63 osteoblast-like cells on the porous surface of nHA/PA66 membrane was better than the bioresponse on the opposite surface of e-PTFE membrane. Because of a better cell attachment manner, there is a potential utilization of the guided bone regeneration (GBR) membrane to substitute nHA/PA66 membrane for e-PTFE membra0008.

  6. Complications of ultrasound-guided percutaneous microwave ablation of renal cell carcinoma

    Science.gov (United States)

    Dong, Xuejuan; Li, Xin; Yu, Jie; Yu, Ming-an; Yu, Xiaoling; Liang, Ping

    2016-01-01

    Purpose To retrospectively review the complications of ultrasound (US)-guided percutaneous microwave ablation (MWA) of renal cell carcinoma. Patients and methods In this study, 101 patients with 105 tumors seen from April 2006 to Feb 2014 were enrolled retrospectively. The patients were treated with US-guided percutaneous MWA and were followed up with contrast-enhanced US and computed tomography or magnetic resonance imaging at 1, 3, and 6 months and every 6 months thereafter. Results Technical success was achieved in 99 of 105 tumors (94.3%). The median follow-up time was 25 (range 1.13–93.23) months. Among the 105 tumors, 26 complications in 24.8% of patients and 23 minor complications (Clavien–Dindo Grades I and II) in 21.9% of patients were noted, accounting for 88.5% of all complications. All the minor complications were cured. Three major complications (Clavien–Dindo Grade ≥III) occurred in 2.9% of the patients, accounting for 11.5% of all complications: hydrothorax in two patients and bowel injury in one. The two patients who had hydrothorax post-MWA had a history of cirrhosis and were treated with catheter drainage. The bowel injury was treated surgically. In all patients, the changes in serum creatinine and urea nitrogen levels from before to after the procedure were small. Conclusion US-guided percutaneous MWA is a beneficial treatment for renal cell carcinoma in selected patients; however, if the renal tumor is close to the bowel, or the patient has serious comorbidities or has undergone abdominal surgery, the procedure must be performed more carefully. PMID:27713644

  7. Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

    OpenAIRE

    Takeda, S; Zou, Y R; Bluethmann, H; Kitamura, D; Muller, U.; Rajewsky, K

    1993-01-01

    Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replac...

  8. Simulation of the PEM fuel cell hybrid power train of an automated guided vehicle and comparison with experimental results

    NARCIS (Netherlands)

    Veenhuizen, Bram; Bosma, J.C.N.

    2009-01-01

    At HAN University research has been started into the development of a PEM fuel cell hybrid power train to be used in an automated guided vehicle. For this purpose a test facility is used with the possibility to test all important functional aspects of a PEM fuel cell hybrid power train. In this pape

  9. Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

    Science.gov (United States)

    Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P; Swayze, Eric E; Bennett, C Frank; Cleveland, Don W

    2015-12-22

    Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

  10. Dynamics of genome rearrangement in bacterial populations.

    Directory of Open Access Journals (Sweden)

    Aaron E Darling

    Full Text Available Genome structure variation has profound impacts on phenotype in organisms ranging from microbes to humans, yet little is known about how natural selection acts on genome arrangement. Pathogenic bacteria such as Yersinia pestis, which causes bubonic and pneumonic plague, often exhibit a high degree of genomic rearrangement. The recent availability of several Yersinia genomes offers an unprecedented opportunity to study the evolution of genome structure and arrangement. We introduce a set of statistical methods to study patterns of rearrangement in circular chromosomes and apply them to the Yersinia. We constructed a multiple alignment of eight Yersinia genomes using Mauve software to identify 78 conserved segments that are internally free from genome rearrangement. Based on the alignment, we applied Bayesian statistical methods to infer the phylogenetic inversion history of Yersinia. The sampling of genome arrangement reconstructions contains seven parsimonious tree topologies, each having different histories of 79 inversions. Topologies with a greater number of inversions also exist, but were sampled less frequently. The inversion phylogenies agree with results suggested by SNP patterns. We then analyzed reconstructed inversion histories to identify patterns of rearrangement. We confirm an over-representation of "symmetric inversions"-inversions with endpoints that are equally distant from the origin of chromosomal replication. Ancestral genome arrangements demonstrate moderate preference for replichore balance in Yersinia. We found that all inversions are shorter than expected under a neutral model, whereas inversions acting within a single replichore are much shorter than expected. We also found evidence for a canonical configuration of the origin and terminus of replication. Finally, breakpoint reuse analysis reveals that inversions with endpoints proximal to the origin of DNA replication are nearly three times more frequent. Our findings

  11. SU-E-I-39: Molecular Image Guided Cancer Stem Cells Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Abdollahi, H

    2014-06-01

    Purpose: Cancer stem cells resistance to radiation is a problematic issue that has caused a big fail in cancer treatment. Methods: As a primary work, molecular imaging can indicate the main mechanisms of radiation resistance of cancer stem cells. By developing and commissioning new probes and nanomolecules and biomarkers, radiation scientist will able to identify the essential pathways of radiation resistance of cancer stem cells. As the second solution, molecular imaging is a best way to find biological target volume and delineate cancer stem cell tissues. In the other hand, by molecular imaging techniques one can image the treatment response in tumor and also in normal tissue. In this issue, the response of cancer stem cells to radiation during therapy course can be imaged, also the main mechanisms of radiation resistance and finding the best radiation modifiers (sensitizers) can be achieved by molecular imaging modalities. In adaptive radiotherapy the molecular imaging plays a vital role to have higher tumor control probability by delivering high radiation doses to cancer stem cells in any time of treatment. The outcome of a feasible treatment is dependent to high cancer stem cells response to radiation and removing all of which, so a good imaging modality can show this issue and preventing of tumor recurrence and metastasis. Results: Our results are dependent to use of molecular imaging as a new modality in the clinic. We propose molecular imaging as a new radiobiological technique to solve radiation therapy problems due to cancer stem cells. Conclusion: Molecular imaging guided cancer stem cell diagnosis and therapy is a new approach in the field of cancer treatment. This new radiobiological imaging technique should be developed in all clinics as a feasible tool that is more biological than physical imaging.

  12. Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure.

    Science.gov (United States)

    Tolosa, Ezequiel J; Fernández-Zapico, Martín E; Battiato, Natalia L; Rovasio, Roberto A

    2016-01-01

    Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

  13. Muscle Interstitial Cells: A Brief Field Guide to Non-satellite Cell Populations in Skeletal Muscle.

    Science.gov (United States)

    Tedesco, Francesco Saverio; Moyle, Louise A; Perdiguero, Eusebio

    2017-01-01

    Skeletal muscle regeneration is mainly enabled by a population of adult stem cells known as satellite cells. Satellite cells have been shown to be indispensable for adult skeletal muscle repair and regeneration. In the last two decades, other stem/progenitor cell populations resident in the skeletal muscle interstitium have been identified as "collaborators" of satellite cells during regeneration. They also appear to have a key role in replacing skeletal muscle with adipose, fibrous, or bone tissue in pathological conditions. Here, we review the role and known functions of these different interstitial skeletal muscle cell types and discuss their role in skeletal muscle tissue homeostasis, regeneration, and disease, including their therapeutic potential for cell transplantation protocols.

  14. Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

    Science.gov (United States)

    Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

    2014-09-01

    Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration.

  15. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia.

    Science.gov (United States)

    Gröschel, Stefan; Sanders, Mathijs A; Hoogenboezem, Remco; de Wit, Elzo; Bouwman, Britta A M; Erpelinck, Claudia; van der Velden, Vincent H J; Havermans, Marije; Avellino, Roberto; van Lom, Kirsten; Rombouts, Elwin J; van Duin, Mark; Döhner, Konstanze; Beverloo, H Berna; Bradner, James E; Döhner, Hartmut; Löwenberg, Bob; Valk, Peter J M; Bindels, Eric M J; de Laat, Wouter; Delwel, Ruud

    2014-04-10

    Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

  16. Chromosomal Rearrangements in Post-Chernobyl Papillary Thyroid Carcinomas: Evaluation by Spectral Karyotyping and Automated Interphase FISH

    Directory of Open Access Journals (Sweden)

    Ludwig Hieber

    2011-01-01

    Full Text Available Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY. In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22 of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87% indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis.

  17. Rearrangements of immunoglobulin genes during differentiation and evolution.

    Science.gov (United States)

    Honjo, T; Nakai, S; Nishida, Y; Kataoka, T; Yamawaki-Kataoka, Y; Takahashi, N; Obata, M; Shimizu, A; Yaoita, Y; Nikaido, T; Ishida, N

    1981-01-01

    Immunoglobulin genes are shown to undergo dynamic rearrangements during differentiation as well as evolution. We have demonstrated that a complete immunoglobulin heavy chain gene is formed by at least two types of DNA rearrangement during B cell differentiation. The first type of rearrangement is V-D-J recombination to complete a variable region sequence and the second type is S-S recombination to switch a constant region sequence. Both types of recombination are accompanied by deletion of the intervening DNA segment. Structure and organization of CH genes are elucidated by molecular cloning and nucleotide sequence determination. Organization of H chain genes is summarized as VH-(unknown distance)-JH-(6.5 kb)-C mu-(4.5 kb)-C delta-(unknown distance)-C gamma 3-(34 kb)-C gamma 1-(21 kb)-C gamma 2b-(15 kb)-C gamma 2a-(14.5 kb)-C epsilon-(12.5 kb)-C alpha. The S-S recombination takes place at the S region which is located at the 5' side of each CH gene. Nucleotide sequence of the S region comprises tandem repetition of closely related sequences. The S-S recombination seems to be mediated by short common sequences shared among S regions. A sister chromatid exchange model was proposed as a mechanism for S-S recombination. Comparison of nucleotide sequences of CH genes indicates that immunoglobulin genes have scrambled by intervening sequence-mediated domain transfer during their evolution.

  18. Comparative study between primary mediastinal B-cell lymphoma and non-mediastinal diffuse large B-cell lymphoma by immunoglobulin gene rearrangement and Epstein-Barr virus infection detection%原发性纵隔B细胞淋巴瘤与非纵隔弥漫性大B细胞淋巴瘤在基因重排检测与EB病毒感染方面的对比研究

    Institute of Scientific and Technical Information of China (English)

    钟定荣; 凌庆; 师晓华; 梁智勇; 刘彤华

    2012-01-01

    Objective To investigate the differences between primary mediastinal B-cell lymphoma (PMBCL) and non-mediastinal conventional diffuse large B-cell common lymphoma (DLBCL) in immunoglobulin gene rearrangement and EB virus infections.Methods Twenty cases of PMBCL and 30 cases of non-mediastinal DLBCL were collected from September,2000 to May,2011.Pathological data were retrospectively analysed.Immunoglobulin heavy chain and light chain gene rearrangements and EBER in-situ hybridization were performed.Results Six of 20 cases of PMBCL showed monoclonal gene rearrangement,all of which were weakly detected.Twenty-seven of 30 cases of ordinary diffuse large B-cell lymphoma showed monoclonal gene rearrangement,which were strongly detected ( 90.0% ).Only 1 of 20 cases PMBCL and 2 of 30 cases of DLBCL were positive for EBER in-situ hybridization.Conclusions The detection rate of immunoglobulin gene rearrangement is significantly lower in PMBCL than that of non-mediastinal DLBCL.However,EB virus infection rates are very low in both types of lymphomas.%目的 探讨原发性纵隔B细胞淋巴瘤(PMBCL)与非纵隔弥漫性大B细胞淋巴瘤(DLBCL)在基因重排检测与EB病毒感染方面的差异.方法 收集2000年9月至2011年5月北京协和医院手术切除的PMBCL 20例,选取同期非纵隔DLBCL 30例作为对照,进行回顾性病理学分析并进行重链和轻链基因重排检测、EBER原位杂交检测.结果 20例PMBCL中6例基因重排检测呈单克隆性,均为弱阳性条带;30例DLBCL中27例基因重排检测呈单克隆性(90.0%),均为强阳性条带;EBER在20例PMBCL中检测到1例阳性,在30例DLBCL中检测到2例阳性.结论 PMBCL在重链和轻链的基因重排检测方面单克隆率低,而非纵隔DLBCL单克隆率高,二者具有显著差异;但原位杂交检测EBER,二者感染率均低,无明显区别.

  19. Molecular screening of pituitary adenomas for gene mutations and rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Herman, V.; Drazin, N.Z.; Gonskey, R.; Melmed, S. (Cedars-Sinai Medical Center, Los Angeles, CA (United States))

    1993-07-01

    Although pituitary tumors arise as benign monoclonal neoplasms, genetic alterations have not readily been identified in these adenomas. The authors studied restriction fragment abnormalities involving the GH gene locus, and mutations in the p53 and H-, K-, and N-ras genes in 22 human GH cell adenomas. Twenty two nonsecretory adenomas were also examined for p53 and ras gene mutations. Seven prolactinoma DNA samples were tested for deletions in the multiple endocrine neoplasia-1 (MEN-1) locus, as well as for rearrangements in the hst gene, a member of the fibroblast growth factor family. In DNA from GH-cell adenomas, identical GH restriction patterns were detected in both pituitary and lymphocyte DNA in all patients and in one patient with a mixed GH-TSH cell adenoma. Using polymerase chain reaction (PCR)-single stranded conformation polymorphism analysis, no mutations were detected in exons 5, 6, 7 and 8 of the p53 gene in GH cell adenomas nor in 22 nonsecretory adenomas. Codons 12/13 and 61 of H-ras, K-ras, and N-ras genes were also intact on GH cell adenomas and in nonsecretory adenomas. Site-specific probes for chromosome 11q13 including, PYGM, D11S146, and INT2 were used in 7 sporadic PRL-secreting adenomas to detect deletions of the MEN-1 locus on chromosome 11. One patient was identified with a loss of 11p, and the remaining 6 patients did not demonstrate loss of heterozygosity in the pituitary 11q13 locus, compared to lymphocyte DNA. None of these patients demonstrated hst gene rearrangements which also maps to this locus. These results show that p53 and ras gene mutations are not common events in the pathogenesis of acromegaly and nonsecretory tumors. Although hst gene rearrangements and deletions of 11q13 are not associated with sporadic PRl-cell adenoma formation, a single patient was detected with a partial loss of chromosome 11, including the putative MEN-1 site. 31 refs., 5 figs., 2 tabs.

  20. Gyrokinetic and kinetic particle-in-cell simulations of guide-field reconnection

    Science.gov (United States)

    Munoz Sepulveda, Patricio Alejandro; Büchner, Jörg; Kilian, Patrick; Told, Daniel; Jenko, Frank

    2016-07-01

    Fully kinetic Particle-in-Cell (PIC) simulations of (strong) guide-field reconnection can be computationally very demanding, due to the intrinsic stability and accuracy conditions required by this numerical method. One convenient approach to circumvent this issue is using gyrokinetic theory, an approximation of the Vlasov-Maxwell equations for strongly magnetized plasmas that eliminates the fast gyromotion, and thus reduces the computational cost. Although previous works have started to compare the features of reconnection between both approaches, a complete understanding of the differences is far from being complete. This knowledge is essential to discern the limitations of the gyrokinetic simulations of magnetic reconnection when applied to scenarios with moderate guide fields, such as the Solar corona, in contrast to most of the fusion/laboratory plasmas. We extend a previous work by our group, focused in the differences in the macroscopic flows, by analyzing the heating processes and non-thermal features developed by reconnection between both plasma approximations. We relate these processes by identifying some high-frequency cross-streaming instabilities appearing only in the fully kinetic approach. We characterize the effects of these phenonema such as anisotropic electron heating, beam formation and turbulence under different parameter regimes. And finally, we identify the conditions under which these instabilities tends to become negligible in the fully kinetic model, and thus a comparison with gyrokinetic theory becomes more reliable.

  1. Catalytic synthesis of amides via aldoximes rearrangement.

    Science.gov (United States)

    Crochet, Pascale; Cadierno, Victorio

    2015-02-14

    Amide bond formation reactions are among the most important transformations in organic chemistry because of the widespread occurrence of amides in pharmaceuticals, natural products and biologically active compounds. The Beckmann rearrangement is a well-known method to generate secondary amides from ketoximes. However, under the acidic conditions commonly employed, aldoximes RHC=NOH rarely rearrange into the corresponding primary amides RC(=O)NH2. In recent years, it was demonstrated that this atom-economical transformation can be carried out efficiently and selectively with the help of metal catalysts. Several homogeneous and heterogenous systems have been described. In addition, protocols offering the option to generate the aldoximes in situ from the corresponding aldehydes and hydroxylamine, or even from alcohols, have also been developed, as well as a series of tandem processes allowing the access to N-substituted amide products. In this Feature article a comprehensive overview of the advances achieved in this particular research area is presented.

  2. Conversion of embryonic stem cells into pancreatic beta-cell surrogates guided by ontogeny.

    Science.gov (United States)

    Lees, Justin G; Tuch, Bernard E

    2006-05-01

    Cellular therapies to treat Type 1 diabetes are being devised and the use of human embryonic stem cells (hESCs) offers a solution to the issue of supply, because hESCs can be maintained in a pluripotent state indefinitely. Furthermore, hESCs have advantages in terms of their plasticity and reduced immunogenicity. Several strategies that have so far been investigated indicate that hESCs are capable of differentiating into insulin producing beta-cell surrogates. However the efficiency of the differentiation procedures used is generally quite low and the cell populations derived are often highly heterogenous. A strategy that appears to have long term potential is to design differentiation procedures based on the ontogeny of the beta-cell. The focus of this strategy is to replicate signaling processes that are known to be involved in the maturation of a beta-cell. The earliest pancreatic progenitors found in the developing vertebrate fetus are produced via a process known as gastrulation and form part of the definitive endoderm germ layer. hESCs have recently been differentiated into definitive endoderm with high efficiency using a differentiation procedure that mimics the signaling that occurs during gastrulation and the formation of the definitive endoderm. Subsequent events during pancreas development involve a section of the definitive endoderm forming into pancreatic epithelium, which then branches into the pancreatic mesenchyme to form islet clusters of endocrine cells. A proportion of the endocrine precursor cells within islets develop into insulin producing beta-cells. The challenge currently is to design hESC differentiation procedures that mimic the combined events of these stages of beta-cell development.

  3. A gut-homing, oligoclonal CD4+ T cell population in severe-combined immunodeficient mice expressing a rearranged, transgenic class I-restricted alpha beta T cell receptor

    DEFF Research Database (Denmark)

    Reimann, J; Rudolphi, A; Spiess, S

    1995-01-01

    R alpha T-beta T+ cells were found in gut tissues of the immunodeficient host. Transplanted scid mice developed clinical and histological signs of IBD. An oligoclonal, gut-homing, memory/effector CD4+ CD44+ TcR beta T+ TcR alpha T-T cell subset from leaky tg scid mice thus has a pathogenic potential when...

  4. Vacancy rearrangement processes in multiply ionized atoms

    Energy Technology Data Exchange (ETDEWEB)

    Czarnota, M [Institute of Physics, Swietokrzyska Academy, 25-406 Kielce (Poland); Pajek, M [Institute of Physics, Swietokrzyska Academy, 25-406 Kielce (Poland); Banas, D [Institute of Physics, Swietokrzyska Academy, 25-406 Kielce (Poland); Dousse, J-Cl [Physics Department, University of Fribourg, CH-1700 Fribourg (Switzerland); Maillard, Y-P [Physics Department, University of Fribourg, CH-1700 Fribourg (Switzerland); Mauron, O [Physics Department, University of Fribourg, CH-1700 Fribourg (Switzerland); Raboud, P A [Physics Department, University of Fribourg, CH-1700 Fribourg (Switzerland); Berset, M [Physics Department, University of Fribourg, CH-1700 Fribourg (Switzerland); Hoszowska, J [European Synchrotron Radiation Facility (ESRF), F-38043 Grenoble (France); Slabkowska, K [Faculty of Chemistry, Nicholas Copernicus University, 87-100 Torun (Poland); Polasik, M [Faculty of Chemistry, Nicholas Copernicus University, 87-100 Torun (Poland); Chmielewska, D [Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland); Rzadkiewicz, J [Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland); Sujkowski, Z [Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland)

    2007-03-01

    We demonstrate that in order to interpret the x-ray satellite structure of Pd L{alpha}{sub 1,2}(L{sub 3}M{sub 4,5}) transitions excited by fast O ions, which was measured using a high-resolution von Hamos crystal spectrometer, the vacancy rearrangement processes, taking place prior to the x-ray emission, have to be taken into account. The measured spectra were compared with the predictions of the multi-con.guration Dirac-Fock (MCDF) calculations using the fluorescence and Coster-Kronig yields which were modiffed due to a reduced number of electrons available for relaxation processes and the effect of closing the Coster-Kronig transitions. We demonstrate that the vacancy rearrangement processes can be described in terms of the rearrangement factor, which can be calculated by solving the system of rate equations modelling the flow of vacancies in the multiply ionized atom. By using this factor, the ionization probability at the moment of collision can be extracted from the measured intensity distribution of x-ray satellites. The present results support the independent electron picture of multiple ionization and indicate the importance of use of Dirac-Hartree-Fock wave functions to calculate the ionization probabilities.

  5. Clinical characteristics and prognosis of concurrent positive t(14;18) and myc gene rearrangement ;in diffuse large B cell lymphoma%t(14;18)和 myc 基因重排双阳性弥漫性大 B 细胞淋巴瘤的临床特征和预后

    Institute of Scientific and Technical Information of China (English)

    张宏伟; 白敏; 苏丽萍; 陈振文; 王列样; 贺建霞; 郑玉萍; 韩维娥; 杨斌; 王艳丽; 赵志强

    2016-01-01

    Objective To study the incidence of positive t(14;18) and myc gene rearrangement, and the clinical features and prognosis of concurrent positive t ( 14;18 ) and myc gene rearrangement“ double-hit lymphoma” (DHL) in diffuse large B cell lymphoma.Me thods The positive t(14;18) and myc gene rearrangement in 106 cases of DLBCL were analyzed using interphase fluorescent in situ hybridization ( FISH ) technique. The expression of myc and bcl-2 proteins was determined by immunohistochemistry.The relationship of positive t ( 14;18) and myc gene rearrangement with clinical features, pathogenesis and prognosis for the patients was analyzed.SPSS 16.0 software was used for statistical analysis.Results Among the 106 cases, there were 27 (25.5%) cases with positive t(14;18) and 13 (12.3%) cases with myc gene rearrangement, and 7 cases (6.6%) of DLBCL with concurrent t(14; 18)-positive and myc gene rearrangement.A relationship was observed between positive t ( 14;18 ) and myc gene rearrangement ( P=0.019) .The follow-up data showed that the 7 DHL patients were in age of 528-4 years, the International Prognostic Index (IPI) scores were 3 in two cases, 4 in four cases and 5 in one case, and the ECOG scores were 3 in all the7 cases .Four patients had bone marrow involvement and were combined with leukemia.The survival time ranged from 0.5 to 6 months, with a median survival of 4 months.The univariate analysis showed that B symptom, Ann Arbor stage, ECOG score, LDH level, IPI score, immunophenotype, bcl-2 protein expression, myc protein expression,and myc gene rearrangement were all associated with poor prognosis ( P<0.05 for all) .The multivariate analysis using a COX proportional hazard model confirmed that ECOG score, bcl-2 protein expression, myc protein expression , myc gene rearrangement, and immunophenotype were independent prognostic factors affecting survival ( P<0.05 for all) , among them, the myc gene rearrangement was the strongest prognostic factor ( OR=4.337,P<0

  6. Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

    Directory of Open Access Journals (Sweden)

    Hyun-Kyoung Kim

    Full Text Available BACKGROUND: The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs, which are abundant in solid tumors, can be utilized for identification of rearranged ends. METHOD: As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP microarray method entailing CNB-region refinement by competitor DNA. RESULT: Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9% were identified, and two polymerase chain reaction (PCR-amplifiable rearrangements were obtained in six cases (66.7%. And significantly, TNGS-CNB, with its high positive identification rate (82.6% of PCR-amplifiable rearrangements at candidate sites (19/23, just from filtering of aligned sequences, requires little effort for validation. CONCLUSION: Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

  7. Vapor cell based sodium laser guide star mechanism study lab-bench

    Science.gov (United States)

    Wang, Hongyan; Li, Lihang; Luo, Ruiyao; Li, Lei; Ning, Yu; Xi, Fengjie; Xu, Xiaojun

    2016-07-01

    Sodium laser guide star (LGS) is the key for the success of modern adaptive optics (AO) supported large ground based telescopes, however, for many field applications, Sodium LGS's brightness is still a limited factor. Large amounts of theoretical efforts have been paid to optimize Sodium LGS exciting parameters, that is, to fully discover potential of harsh environment surrounding mesospheric extreme thin sodium atoms under resonant excitation, whether quantum or Monte Carlo based. But till to now, only limited proposals are demonstrated with on-sky test due to the high cost and engineering complexities. To bridge the gap between theoretical modeling and on-sky test, we built a magnetic field controllable sodium cell based lab-bench, which includes a small scale sum-frequency single mode 589nm laser, with added amplitude, polarization, and phase modulators. We could perform quantitative resonant fluorescence study under single, multi-frequency, side-band optical re-pumping exciting with different polarization, also we could perform optical field modulation to study Larmor precession which is considered as one of devils of Sodium LGS, and we have the ability to generate beams contain orbital angular moment. Our preliminary sodium cell based optical re-pumping experiments have shown excellent consistence with Bloch equation predicted results, other experimental results will also be presented in the report, and these results will give a direct support that sodium cell based lab-bench study could help a Sodium LGS scientists a lot before their on-sky test.

  8. Detection and precise mapping of germline rearrangements in BRCA1, BRCA2, MSH2, and MLH1 using zoom-in array comparative genomic hybridization (aCGH)

    DEFF Research Database (Denmark)

    Staaf, Johan; Törngren, Therese; Rambech, Eva;

    2008-01-01

    deletions or duplications occurring in BRCA1 (n=11), BRCA2 (n=2), MSH2 (n=7), or MLH1 (n=9). Additionally, we demonstrate its applicability for uncovering complex somatic rearrangements, exemplified by zoom-in analysis of the PTEN and CDKN2A loci in breast cancer cells. The sizes of rearrangements ranged...

  9. Breaking Good: Accounting for Fragility of Genomic Regions in Rearrangement Distance Estimation.

    Science.gov (United States)

    Biller, Priscila; Guéguen, Laurent; Knibbe, Carole; Tannier, Eric

    2016-01-01

    Models of evolution by genome rearrangements are prone to two types of flaws: One is to ignore the diversity of susceptibility to breakage across genomic regions, and the other is to suppose that susceptibility values are given. Without necessarily supposing their precise localization, we call "solid" the regions that are improbably broken by rearrangements and "fragile" the regions outside solid ones. We propose a model of evolution by inversions where breakage probabilities vary across fragile regions and over time. It contains as a particular case the uniform breakage model on the nucleotidic sequence, where breakage probabilities are proportional to fragile region lengths. This is very different from the frequently used pseudouniform model where all fragile regions have the same probability to break. Estimations of rearrangement distances based on the pseudouniform model completely fail on simulations with the truly uniform model. On pairs of amniote genomes, we show that identifying coding genes with solid regions yields incoherent distance estimations, especially with the pseudouniform model, and to a lesser extent with the truly uniform model. This incoherence is solved when we coestimate the number of fragile regions with the rearrangement distance. The estimated number of fragile regions is surprisingly small, suggesting that a minority of regions are recurrently used by rearrangements. Estimations for several pairs of genomes at different divergence times are in agreement with a slowly evolvable colocalization of active genomic regions in the cell.

  10. MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

    Science.gov (United States)

    Bandopadhayay, Pratiti; Ramkissoon, Lori A; Jain, Payal; Bergthold, Guillaume; Wala, Jeremiah; Zeid, Rhamy; Schumacher, Steven E; Urbanski, Laura; O'Rourke, Ryan; Gibson, William J; Pelton, Kristine; Ramkissoon, Shakti H; Han, Harry J; Zhu, Yuankun; Choudhari, Namrata; Silva, Amanda; Boucher, Katie; Henn, Rosemary E; Kang, Yun Jee; Knoff, David; Paolella, Brenton R; Gladden-Young, Adrianne; Varlet, Pascale; Pages, Melanie; Horowitz, Peleg M; Federation, Alexander; Malkin, Hayley; Tracy, Adam A; Seepo, Sara; Ducar, Matthew; Van Hummelen, Paul; Santi, Mariarita; Buccoliero, Anna Maria; Scagnet, Mirko; Bowers, Daniel C; Giannini, Caterina; Puget, Stephanie; Hawkins, Cynthia; Tabori, Uri; Klekner, Almos; Bognar, Laszlo; Burger, Peter C; Eberhart, Charles; Rodriguez, Fausto J; Hill, D Ashley; Mueller, Sabine; Haas-Kogan, Daphne A; Phillips, Joanna J; Santagata, Sandro; Stiles, Charles D; Bradner, James E; Jabado, Nada; Goren, Alon; Grill, Jacques; Ligon, Azra H; Goumnerova, Liliana; Waanders, Angela J; Storm, Phillip B; Kieran, Mark W; Ligon, Keith L; Beroukhim, Rameen; Resnick, Adam C

    2016-03-01

    Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

  11. 多重PCR法检测初诊成人急性淋巴细胞白血病患者克隆性免疫球蛋白和T细胞受体基因重排%Detection of clonal immunoglobulin and T-cell receptor gene rearrangements in newly diagnosed adult patients with acute lymphoblastic leukemia by using multiplex PCR protocols

    Institute of Scientific and Technical Information of China (English)

    姚利; 陈子兴; 岑建农; 梁建英; 何军; 祁小飞; 沈宏杰

    2008-01-01

    Objective To provide the evidence of RQ-PCR-based assessment of minimal residual disease(MRD), the clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements were identified in newly diagnosed adult patients with acute iymphoblastic leukemia (ALL) by multiplex PCR protocols. Methods Forty newly diagnosed adult patients with B-lineage(B-) and T cell (T-) ALL were involvled in this study. All DNA samples were obtained from the bone marrow (BM) mononuclear cells(MNC). IgH、 IgK ,TCRB,TCRG and TCRD gene rearrangements were detected by BIOMED-2 multiplex PCR protocols, which included 96 different primers and 14 multiplex PCR tubes. Results The clonal immunoglobulin(Ig) rearrangements were found in 96% of B- ALL, 86% being IgH and 14% IgK. While in T-ALL, clonal TCR rearrangements were found in all of the patients, 83% being TCRB ,78% TCRG and 39% TCRD. More than two clonal markers were found in 91% of B- ALL and 89% of T- ALL patients. Conclusions The detection rate of clonal rearrangements using the BIOMED-2 14 multiplex PCR tubes is unprecedentedly high, which can detect virtually all clonal B and T-cell proliferations. It can be used for diagnostic cionality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.%目的 应用多莺PCR方法检测初诊成人急性淋巴细胞白血病(ALL)患者的克隆性免疫球蛋白(Ig)和T细胞受体(TCR)基因重排,为实时定量RT-PCR(RQ-PCR)法监测ALL患者体内的微量残留病(MRD)奠定基础.方法 参照BIOMED-2协作组制定的Ig和(或)TCR检测方法,设计96条不同的PCR引物,分成14个混合管,通过多重PCR,分别检测患者骨髓单个核细胞的IgH、IgK、TCRB、TCRG、TCRD克隆性基因重排.结果 在22例成人B系ALL患者中,Ig克隆性重排检出率为96%,其中IgH为86%,IgK为14%.在18例成人T系ALL患者中,TCR克隆性重排检出率为100%,其中TCRB为83%,TCRG为78%,TCRD为39%.两个及两个以上克隆性

  12. Insights into structural variations and genome rearrangements in prokaryotic genomes.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2015-01-01

    Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing.

  13. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia

    NARCIS (Netherlands)

    Gröschel, Stefan; Sanders, Mathijs A; Hoogenboezem, Remco; de Wit, Elzo; Bouwman, Britta A M; Erpelinck, Claudia; van der Velden, Vincent H J; Havermans, Marije; Avellino, Roberto; van Lom, Kirsten; Rombouts, Elwin J; van Duin, Mark; Döhner, Konstanze; Beverloo, H Berna; Bradner, James E; Döhner, Hartmut; Löwenberg, Bob; Valk, Peter J M; Bindels, Eric M J; de Laat, Wouter; Delwel, Ruud

    2014-01-01

    Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional geno

  14. Contribution of ultrasound-guided fine-needle aspiration cell blocks of metastatic supraclavicular lymph nodes to the diagnosis of lung cancer

    Directory of Open Access Journals (Sweden)

    Hai-Ying Tian

    2015-01-01

    Conclusion: Cell-block samples from US-guided FNA is a promising, relatively noninvasive technique to provide additional information in lung cancer diagnosis. Analysis of cell blocks allows for genetic analysis of the patients with supraclavicular lymph nodes metastasis.

  15. Detection of B-cell Non-Hodgkin lymphoma antigen rearrangement using BIOMED-2 system in paraffin-embedded specimens%BIOMED-2系统应用于B细胞非霍奇金淋巴瘤抗原受体基因重排检测的研究

    Institute of Scientific and Technical Information of China (English)

    黄娟; 刘浩; 曾凡才; 陈明; 刘萍; 莫冬阳; 李鑫

    2015-01-01

    目的:探讨BIOMED-2系统在检测B细胞非霍奇金淋巴瘤中的应用价值.方法:选取石蜡包埋组织B细胞非霍奇金淋巴瘤40例、其他淋巴结增生性病变10例,提取组织DNA,应用BIOMED-2系统中的61条引物分成9个组别进行PCR扩增,检测DNA质量及抗原受体基因重排克隆性.结果:94%(47/50)样本DNA长度在300 bp以上,BIOMED-2系统检测的敏感性和特异性均为100%,IgH-A、IgH-B、IgH-C、IgH-D、IgH-E、IgK-A、IgK-B、IgL组检出率分别为45%(18/40)、27.5%(11/40)、87.5%(35/40)、55%(22/40)、47.5%(19/40)、80%(32/40)、50%(20/40)、22.5%(9/40),IgH-C 联合 IgK-A 组合检出率为100%. 结论:BIOMED-2系统适用于石蜡包埋组织B细胞抗原受体基因重排检测,具有很高的敏感性和特异性.%Objective: To evaluate the BIOMED-2 system in detecting the monoclonality of antigen receptor gene rearrangement in B-cell Non-Hodgkin lymphoma. Methods: Genomic DNA was extracted from paraffin-embedded specimens of 40 cases of B cell Non-Hodgkin lymphoma and 10 cases of proliferative diseases of lymph nodes, and clonality of antigen receptor gene rearrangement was assessed using the 9 groups of total 61 primers of BIOMED-2 system. Results: 94% (47/50) of specimens yielded PCR products over 300 bp using DNA quality control primers. The sensitivity and specificity of BIOMED-2 system in the detection of monoclona-lity of antigen receptor gene rearrangement were both 100%. The rate of detection of monoclonality using primes for IgH-A,IgH-B,IgH-C,IgH-D,IgH-E,IgK-A,IgK-B,IgL was 45%(18/40),27.5%(11/40),87.5%(35/40), 55%(22/40),47.5%(19/40),80%(32/40),50%(20/40) and 22.5%(9/40)respectively. When combining IgH-C and IgK-A primers, all cases of antigen receptor gene monoclonality rearrangement could be detected. Conclusion:BIOMED-2 system is a highly sensitive and specific method in detecting the monoclonality of antigen receptor gene rearrangement of B cell Non-Hodgkin lymphoma in routine paraffin

  16. The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJβ rearrangement.

    Directory of Open Access Journals (Sweden)

    Karen S Hathcock

    Full Text Available T cell development occurs in the thymus and is critically dependent on productive TCRβ rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (β checkpoint, which is uniquely permissive for V-DJβ recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRβ rearrangement and pre-TCR expression enforces suppression of TCRβ rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRβ product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the β checkpoint, would suppress all V-DJβ rearrangement. We previously reported that premature CD28 signaling in murine CD4(-CD8(- (DN thymocytes supports differentiation of CD4(+CD8(+ (DP cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRβ rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRβ rearrangement occurs, V-DJβ rearrangement never occurs, even in DP cells that are permissive for D-Jβ and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJβ suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRβ rearrangement to occur.

  17. Functionalization and cellular uptake of boron carbide nanoparticles. The first step toward T cell-guided boron neutron capture therapy.

    Science.gov (United States)

    Mortensen, M W; Björkdahl, O; Sørensen, P G; Hansen, T; Jensen, M R; Gundersen, H J G; Bjørnholm, T

    2006-01-01

    In this paper we present surface modification strategies of boron carbide nanoparticles, which allow for bioconjugation of the transacting transcriptional activator (TAT) peptide and fluorescent dyes. Coated nanoparticles can be translocated into murine EL4 thymoma cells and B16 F10 malignant melanoma cells in amounts as high as 0.3 wt. % and 1 wt. %, respectively. Neutron irradiation of a test system consisting of untreated B16 cells mixed with B16 cells loaded with boron carbide nanoparticles were found to inhibit the proliferative capacity of untreated cells, showing that cells loaded with boron-containing nanoparticles can hinder the growth of neighboring cells upon neutron irradiation. This could provide the first step toward a T cell-guided boron neutron capture therapy.

  18. Adiabatic Rearrangement of Hollow PV Towers

    Directory of Open Access Journals (Sweden)

    Eric A Hendricks

    2010-10-01

    Full Text Available Diabatic heating from deep moist convection in the hurricane eyewall produces a towering annular structure of elevated potential vorticity (PV. This structure has been referred to as a hollow PV tower. The sign reversal of the radial gradient of PV satisfies the Charney-Stern necessary condition for combined barotropic-baroclinic instability. For thin enough annular structures, small perturbations grow exponentially, extract energy from the mean flow, and lead to hollow tower breakdown, with significant vortex structural and intensity change. The three-dimensional adiabatic rearrangements of two prototypical hurricane-like hollow PV towers (one thick and one thin are examined in an idealized framework. For both hollow towers, dynamic instability causes air parcels with high PV to be mixed into the eye preferentially at lower levels, where unstable PV wave growth rates are the largest. Little or no mixing is found to occur at upper levels. The mixing at lower and middle levels is most rapid for the breakdown of the thin hollow tower, consistent with previous barotropic results. For both hollow towers, this advective rearrangement of PV affects the tropical cyclone structure and intensity in a number of ways. First, the minimum central pressure and maximum azimuthal mean velocity simultaneously decrease, consistent with previous barotropic results. Secondly, isosurfaces of absolute angular momentum preferentially shift inward at low levels, implying an adiabatic mechanism by which hurricane eyewall tilt can form. Thirdly, a PV bridge, similar to that previously found in full-physics hurricane simulations, develops as a result of mixing at the isentropic levels where unstable PV waves grow most rapidly. Finally, the balanced mass field resulting from the PV rearrangement is warmer in the eye between 900 and 700 hPa. The location of this warming is consistent with observed warm anomalies in the eye, indicating that in certain instances the hurricane

  19. Vacancy rearrangement processes in multiply ionized atoms

    OpenAIRE

    Czarnota, M.; Pajek, M.; Banas, D.; Dousse, Jean-Claude; Maillard, Yves-Patrick; Mauron, Olivier; Raboud, Pierre-Alexandre; Berset, Michel; Hoszowska, J.; Slabkowska, K.; Polasik, M.; Chmielewska, D; Rzadkiewicz, J.; Sujkowski, Z.

    2007-01-01

    We demonstrate that in order to interpret the x-ray satellite structure of Pd Lα1,2(L₃M4,5) transitions excited by fast O ions, which was measured using a high-resolution von Hamos crystal spectrometer, the vacancy rearrangement processes, taking place prior to the x-ray emission, have to be taken into account. The measured spectra were compared with the predictions of the multi-con.guration Dirac-Fock (MCDF) calculations using the fluorescence and Coster-Kronig yields which were modiffed due...

  20. Bcl-2 GENE REARRANGEMENT DETERMINED BY PCR AS A MEAN TO DETECT MINIMAL RESIDUAL DISEASE IN MALIGNANT LYMPHOMAS

    Institute of Scientific and Technical Information of China (English)

    XIANG Zhi-fu; LU Yu-ying; LAI Yong-rong; CHEN Yan; LI Hui-yu; ZOU Ping

    1999-01-01

    Objective: To develop a sensitive method to detect minimal residual disease and to elucidate the significance of bcl-2 gene rearrangement in diagnosis and treatment of malignant lymphoma. Methods: Using polymerase chain reaction (PCR) to detect bcl-2 gene rearrangement and using serial dilution method to define the sensitivity of PCR. Results: In 9 different malignant lymphoma cell lines, Su-DHL-4 and Su-DHL-6 were shown bcl-2(MBR)/JH rearrangement, the sensitivity of PCR was 1:105. In 16 patients with follicular lymphoma, the peripheral blood and bone marrow were PCR positive in 4 cases both at initial diagnosis and after complete remission. Conclusion:Detection of bcl-2 gene rearrangement by PCR provides a sensitive and specific assay of minimal residual disease.It is helpful to improve staging of disease, prognosis and evaluation of the treatment results.

  1. Limited pattern of TCR delta chain gene rearrangement on the RNA level in multiple sclerosis.

    Science.gov (United States)

    Nowak, J; Januszkiewicz, D; Pernak, M; Hertmanowska, H; Nowicka-Kujawska, K; Rembowska, J; Lewandowski, K; Nowak, T; Wender, M

    2001-01-01

    Susceptibility to multiple sclerosis (MS) is most likely affected by a number of genes, including HLA and T-cell receptor (TCR) genes. T cells expressing gamma/delta receptors seem to contribute to autoagression in MS, as evidenced by their localization in the MS plaques in the brain. The aim of this study was to analyse the TCRdelta chain gene rearrangement at the RNA (cDNA) level and compare to the DNA pattern rearrangement. TCRdelta gene rearrangement was analysed in MS patients and healthy individuals with the use of primers specific for Vdelta1-6 and Jdelta1 genes (at the DNA level) and specific for Vdelta1-6 and Cdelta1 genes (at the cDNA level). The size of PCR products was analysed on agarose gel and by ALF-Express (Pharmacia). Additionally, the lymphocyte surface immunophenotype was studied with specific monoclonal antibodies. At the DNA level a restricted pattern of Vdelta3-Jdelta1 and Vdelta5-Jdelta1 was found only in MS patients. Contrary to DNA, mono-, oligoclonal RNA (cDNA) rearrangements were limited to Vdelta1-Cdelta1, Vdelta2-Cdelta1 and Vdelta3-Cdelta1 only in MS patients as well. Surface immunophenotype analysis revealed in MS a much higher frequency of activated gamma/delta T lymphocytes, i.e. expressing HLA-DR and CD25. An elevated level of CD56 positive cells in MS was recorded. Mono-oligoclonal pattern of TCRdelta gene rearrangement at the RNA level, along with increase in activated gamma/delta T cells, strongly argue for a significant role of gamma/delta T lymphocytes in the pathogenesis of MS.

  2. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.

    Science.gov (United States)

    Kim, Sojung; Kim, Daesik; Cho, Seung Woo; Kim, Jungeun; Kim, Jin-Soo

    2014-06-01

    RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

  3. Assessment of BIOMED-2 assays for detection of clonal Ig gene rearrangements in mature B-cell lymphomas%BIOMED-2聚合酶链反应Ig基因重排对成熟非霍奇金B细胞淋巴瘤诊断的价值

    Institute of Scientific and Technical Information of China (English)

    张婧; 吴迎辉; 孔海鹰; 周小鸽; 金哈斯; 吴晓明; 张丹丹; 宫丽平

    2009-01-01

    目的 探讨BIOMED-2聚合酶链反应(PCR)在成熟非霍奇金B细胞淋巴瘤(B-NHL)诊断中的价值.方法 收集成熟B-NHL组织标本72例,其中弥漫性大B细胞淋巴瘤37例,黏膜相关淋巴组织结外边缘区淋巴瘤35例为研究对象,并以反应性增生病变25例作为对照.提取以上组织的DNA,并以PCR来检测其完整性和可扩增性,选取质量合格的DNA.85.6%(83/97)的样品DNA长度>300 bp,其中60例成熟B-NHL和23例反应性增生可用于BIOMED-2 PCR检测免疫球蛋白重链(IgH)和kappa轻链(IgK)基因重排的克隆性.结果 利用BIOMED-2 PCR检测的60例成熟B-NHL中,57例存在Ig基因的克隆性重排,其检测敏感性为95%,23例反应性增生病例中未出现Ig基因的克隆性重排,其检测特异性为100%.结论 BIOMED-2 PCR适用于石蜡包埋组织.该方法具有很高的敏感性和特异性,对成熟B-NHL诊断的辅助价值很高.%Objective To evaluate the efficiency of the BIOMED-2 PCR assay and its implication in the diagnosis of mature B-cell non-Hodgkin's lymphomas. Methods Clinical, morphological and immunohistochemical features of 72 cases of non-Hodgkin's lymphomas were studied, including 25 reactive lymphoid hyperplasia, 37 diffuse large B cell lymphomas (DLBCL) and 35 extranodal marginal zone lymphomas of mucosa associated lymphoid tissues (MALT lymphoma and in addition, 25 cases of reactive lymphoid hyperplasia were used as the controls). DNA was exacted from the paraffin embedded formalin fixed tissue blocks and the quality of DNA was assessed using the BIOMED-2 specimen control reaction. Adequate samples were then analyzed by BIOMED-2 for immunoglobulin heavy and kappa light chain rearrangements. Results Adequate DNA was obtained in 83 of 97 samples, including 60 mature B cell lymphomas and 23 reactive lymphoid hyperplasia. Clonal B-cell gene rearrangements were detected in 57 of 60(95%) lymphomas. In contrast, clonal Ig gene rearrangements were not detected in any of the 23

  4. Presence of interleukin-4-producing cells for human bone regeneration after application of guided tissue regeneration membranes.

    Science.gov (United States)

    Kabashima, H; Nagata, K

    2001-07-01

    To study the process of bone regeneration we examined three samples of periapical regenerative tissue obtained from two patients under a guided tissue regeneration treatment in endodontic surgery by the immunohistochemical and enzyme histochemical methods. The regenerative tissue consisted of a large number of fibroblast-like cells and a small number of mononuclear cells. Fibroblast-like cells stained positively for alkaline phosphatase and osteopontin, whereas mononuclear cells stained positively for CD4. Interleukin-4-producing cells could be detected in adjacent sections. However, interferon-y-producing cells could not be detected. These findings suggest that interleukin-4-producing cells may be one of the elements associated with success in the human bone regeneration process in vivo.

  5. Chromosomal rearrangement interferes with meiotic X chromosome inactivation

    OpenAIRE

    Homolka, David; Ivanek, Robert; Capkova, Jana; Jansa, Petr; Forejt, Jiri

    2007-01-01

    Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by the incomplete meiotic synapsis of rearranged chromosomes, by their colocalization with the XY body in primary spermatocytes, and by male-limited sterility. Previously, we argued that such X–autosomal associations could interfere with meiotic sex chromosome inactivation. Recently, supporting evidence has reported modifications of histones in rearranged chromosomes by a process called the meiotic silencin...

  6. Sterile DJH rearrangements reveal that distance between gene segments on the human Ig H chain locus influences their ability to rearrange

    DEFF Research Database (Denmark)

    Hansen, Tina Østergaard; Lange, Anders Blaabjerg; Barington, Torben

    2015-01-01

    Rearrangement of the Ig locus occurs in two steps. First, a JH gene is rearranged to a D gene followed by a VH gene rearranging to the DJH rearrangement. By next generation sequencing, we analyzed 9969 unique DJH rearrangements and 5919 unique VHDJH rearrangements obtained from peripheral blood B...... frequently than JH locus distal D genes, whereas VH locus proximal D genes were observed more frequently in nonproductive VHDJH rearrangements. We further demonstrate that the distance between VH, D, and JH gene segments influence their ability to rearrange within the human Ig locus....

  7. Clinical and cytogenetic features of a population-based consecutive series of 285 pediatric T-cell acute lymphoblastic leukemias: rare T-cell receptor gene rearrangements are associated with poor outcome

    DEFF Research Database (Denmark)

    Karrman, Kristina; Forestier, Erik; Heyman, Mats

    2009-01-01

    Clinical characteristics and cytogenetic aberrations were ascertained and reviewed in a population-based consecutive series of 285 pediatric T-cell acute lymphoblastic leukemias (T-ALLs) diagnosed between 1992 and 2006 in the Nordic countries. Informative karyotypic results were obtained in 249 (87...... incidence of mediastinal mass and central nervous system involvement (59% and 9.5%, respectively). These features did not differ significantly among the various genetic subgroups. 5-year event-free survival (EFS) and overall survival for all patients were 0.61 (+/-0.03) and 0.67 (+/-0.03), respectively....... In a multivariate analysis, two factors affected negatively the EFS, namely a WBC count of > or =200 x 10(9)/l (P

  8. Applications of the Wittig-Still rearrangement in organic synthesis.

    Science.gov (United States)

    Rycek, Lukas; Hudlicky, Tomas

    2017-02-16

    This review traces the discovery of the Wittig-Still rearrangement and its applications in organic synthesis. Its relationship to Wittig rearrangements is discussed along with detailed analysis of E/Z- and diastereoselectivity. Modifications of the products arising from the Wittig-Still rearrangement are reviewed in the context of increased complexity in intermediates potentially useful in target oriented synthesis. Early applications of the Wittig-Still rearrangement to modifications of steroids are reviewed as are applications to various terpene and alkaloid natural product targets and miscellaneous compounds. To the best of our knowledge, the literature is covered through December 2016.

  9. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology.

    Science.gov (United States)

    Blasco, Rafael B; Karaca, Elif; Ambrogio, Chiara; Cheong, Taek-Chin; Karayol, Emre; Minero, Valerio G; Voena, Claudia; Chiarle, Roberto

    2014-11-20

    Generation of genetically engineered mouse models (GEMMs) for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs). Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk-rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo.

  10. 增强免疫组化和原位杂交方法检测非小细胞肺癌的ALK重排的临床可行性%Assessment of ALK Rearrangement in Non-small Cell Lung Cancer:Using Enhancing Immunohistochemical Way and Fluorescence in situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    孟辉; 高献争; 张岚; 刘芳; 李文才

    2015-01-01

    Background and objective Besides epidermal growth factor receptor (EGFR) mutation, the non-small cell lung cancer (NSCLC) of anaplastic lymphoma kinase (ALK) rearrangement becomes another important clinical subtype. A speciifc and high-sensitive and economical detection way is convenience for identiifcation of ALK positive NSCLC quickly and accurately. So the objective of our research is to detect ALK rearrangement in 172 cases of NSCLC by using enhancing immunohistochemical way (ventana-IHC, V-IHC). Methods ALK rearrangement in 172 NSCLC samples was detected by us-ing V-IHC, and positive staining cases were further veriifed by lfuorescence in situ hybridization (FISH). Results Among 172 NSCLC cases, there were 12 positive staining. hTe positive results were conifrmed by FISH and 11 cases were FISH positive. hTe overall concordance between V-IHC and FISH is 91.7%(11/12). Conclusion hTe V-IHC method is a reliable method for ALK arrangement and could be used in clinical screen and diagnosis.%背景与目的继表皮生长因子(epidermal growth factor receptor, EGFR)突变之后,间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)基因重排的非小细胞肺癌(non-small cell lung cancer, NSCLC)已经成为了肺癌的又一重要的临床分型。在临床上需要选择一种特异,灵敏并且价廉的方法准确快速地找到ALK阳性的NSCLC患者。为此本研究探讨增强免疫组化法(ventana-IHC, V-IHC)检测ALK重排的临床可行性。方法利用V-IHC检测172例NSCLC患者ALK重排,阳性患者以荧光原位杂交法(lfuorescence in situ hybridization, FISH)验证。结果172例NSCLC患者中有12例为ALK阳性,经过FISH验证,11例患者为阳性,符合率为91.7%。结论在NSCLC中,V-IHC是ALK检测切实可行的方法,适用于ALK重排的NSCLC的筛查和诊断。

  11. Electric fields guide migration of epidermal stem cells and promote skin wound healing.

    Science.gov (United States)

    Li, Li; Gu, Wei; Du, Juan; Reid, Brian; Deng, Xianjian; Liu, Zhidai; Zong, Zhaowen; Wang, Haiyan; Yao, Bo; Yang, Ce; Yan, Jun; Zeng, Ling; Chalmers, Laura; Zhao, Min; Jiang, Jianxin

    2012-01-01

    Migration of epidermal stem cells (EpSCs) into wounds may play an important role in wound healing. Endogenous electric fields (EFs) arise naturally at wounds. Consistent with previous reports, we measured outward electric currents at rat skin wounds using vibrating probes. Topical use of prostaglandin E2 significantly promoted wound healing. However, it is not known whether EpSCs respond to EFs. We first isolated and characterized EpSCs from rat skin. We then demonstrated that EpSCs isolated from the epidermis migrated directionally toward the cathode in EFs of 50-400 mV/mm. The directedness values increased in a dose- and time-dependent fashion. The migration speed of EpSCs was significantly increased in EFs. EFs induced asymmetric polymerization of intracellular F-actin and activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase (PI3K)/protein kinase B pathways. Inhibition of epidermal growth factor receptor, extracellular signal-regulated kinase 1/2, or PI3K significantly inhibited the cathodal distribution of F-actin and the electrotactic response of EpSCs. These data for the first time show that EpSCs possess obvious electrotaxis, in which the epidermal growth factor receptor-mitogen activated protein kinase-PI3K pathways are involved. These data thus suggest a novel aspect of electric signaling in wound healing-to stimulate and guide migration of EpSCs and to regulate wound healing.

  12. Confocal Microscopy–Guided Laser Ablation for Superficial and Early Nodular Basal Cell Carcinoma

    Science.gov (United States)

    Chen, Chih-Shan Jason; Sierra, Heidy; Cordova, Miguel; Rajadhyaksha, Milind

    2014-01-01

    Importance Laser ablation is a rapid and minimally invasive approach for the treatment of superficial skin cancers, but efficacy and reliability vary owing to lack of histologic margin control. High-resolution reflectance confocal microscopy (RCM) may offer a means for examining margins directly on the patient. Observations We report successful elimination of superficial and early nodular basal cell carcinoma (BCC) in 2 cases-, using RCM imaging to guide Er-:YAG laser ablation. Three-dimensional (3-D) mapping is feasible with RCM-, to delineate the lateral border and thickness of the tumor. Thus, the surgeon may deliver laser fluence and passes with localized control—ie, by varying the ablation parameters in sub-lesional areas with specificity that is governed by the 3-D topography of the BCC. We further demonstrate intra-operative detection of residual BCC after initial laser ablation and complete removal of remaining tumor by additional passes. Both RCM imaging and histologic sections confirm the final clearance of BCC. Conclusions and Relevance Confocal microscopy may enhance the efficacy and reliability of laser tumor ablation. This report represents a new translational application for RCM imaging, which, when combined with an ablative laser, may one day provide an efficient and cost-effective treatment for BCC. PMID:24827701

  13. Sodium vapor cell laser guide star experiments for continuous wave model validation

    Science.gov (United States)

    Pedreros Bustos, Felipe; Holzlöhner, Ronald; Budker, Dmitry; Lewis, Steffan; Rochester, Simon

    2016-07-01

    Recent numerical simulations and experiments on sodium Laser Guide Star (LGS) have shown that a continuous wave (CW) laser with circular polarization and re-pumping should maximize the fluorescent photon return flux to the wavefront sensor for adaptive optics applications. The orientation and strength of the geomagnetic field in the sodium layer also play an important role affecting the LGS return ux. Field measurements of the LGS return flux show agreement with the CW LGS model, however, fluctuations in the sodium column abundance and geomagnetic field intensity, as well as atmospheric turbulence, induce experimental uncertainties. We describe a laboratory experiment to measure the photon return flux from a sodium vapor cell illuminated with a 589 nm CW laser beam, designed to approximately emulate a LGS under controlled conditions. Return flux measurements are carried out controlling polarization, power density, re-pumping, laser linewidth, and magnetic field intensity and orientation. Comparison with the numerical CW simulation package Atomic Density Matrix are presented and discussed.

  14. Evaluation of biodegradable electric conductive tube-guides and mesenchymal stem cells

    Science.gov (United States)

    Ribeiro, Jorge; Pereira, Tiago; Caseiro, Ana Rita; Armada-da-Silva, Paulo; Pires, Isabel; Prada, Justina; Amorim, Irina; Amado, Sandra; França, Miguel; Gonçalves, Carolina; Lopes, Maria Ascensão; Santos, José Domingos; Silva, Dina Morais; Geuna, Stefano; Luís, Ana Lúcia; Maurício, Ana Colette

    2015-01-01

    AIM: To study the therapeutic effect of three tube-guides with electrical conductivity associated to mesenchymal stem cells (MSCs) on neuro-muscular regeneration after neurotmesis. METHODS: Rats with 10-mm gap nerve injury were tested using polyvinyl alcohol (PVA), PVA-carbon nanotubes (CNTs) and MSCs, and PVA-polypyrrole (PPy). The regenerated nerves and tibialis anterior muscles were processed for stereological studies after 20 wk. The functional recovery was assessed serially for gait biomechanical analysis, by extensor postural thrust, sciatic functional index and static sciatic functional index (SSI), and by withdrawal reflex latency (WRL). In vitro studies included cytocompatibility, flow cytometry, reverse transcriptase polymerase chain reaction and karyotype analysis of the MSCs. Histopathology of lung, liver, kidneys, and regional lymph nodes ensured the biomaterials biocompatibility. RESULTS: SSI remained negative throughout and independently from treatment. Differences between treted groups in the severity of changes in WRL existed, showing a faster regeneration for PVA-CNTs-MSCs (P < 0.05). At toe-off, less acute ankle joint angles were seen for PVA-CNTs-MSCs group (P = 0.051) suggesting improved ankle muscles function during the push off phase of the gait cycle. In PVA-PPy and PVA-CNTs groups, there was a 25% and 42% increase of average fiber area and a 13% and 21% increase of the “minimal Feret’s diameter” respectively. Stereological analysis disclosed a significantly (P < 0.05) increased myelin thickness (M), ratio myelin thickness/axon diameter (M/d) and ratio axon diameter/fiber diameter (d/D; g-ratio) in PVA-CNT-MSCs group (P < 0.05). CONCLUSION: Results revealed that treatment with MSCs and PVA-CNTs tube-guides induced better nerve fiber regeneration. Functional and kinematics analysis revealed positive synergistic effects brought by MSCs and PVA-CNTs. The PVA-CNTs and PVA-PPy are promising scaffolds with electric conductive properties, bio

  15. Androgen receptor gene mutation, rearrangement, polymorphism.

    Science.gov (United States)

    Eisermann, Kurtis; Wang, Dan; Jing, Yifeng; Pascal, Laura E; Wang, Zhou

    2013-09-01

    Genetic aberrations of the androgen receptor (AR) caused by mutations, rearrangements, and polymorphisms result in a mutant receptor that has varied functions compared to wild type AR. To date, over 1,000 mutations have been reported in the AR with most of these being associated with androgen insensitivity syndrome (AIS). While mutations of AR associated with prostate cancer occur less often in early stage localized disease, mutations in castration-resistant prostate cancer (CRPC) patients treated with anti-androgens occur more frequently with 10-30% of these patients having some form of mutation in the AR. Resistance to anti-androgen therapy usually results from gain-of-function mutations in the LBD such as is seen with bicalutamide and more recently with enzalutamide (MDV3100). Thus, it is crucial to investigate these new AR mutations arising from drug resistance to anti-androgens and other small molecule pharmacological agents.

  16. Level rearrangement in three-body systems

    CERN Document Server

    Richard, Jean-Marc

    2016-01-01

    We study systems of three bosons bound by a long-range interaction supplemented by a short-range potential of variable strength. This generalizes the usual two-body exotic atoms where the Coulomb interaction is modified by nuclear forces at short distances. The energy shift due to the short-range part of the interaction combines two-body terms similar to the ones entering the Trueman-Deser formula, and three-body contributions. A phenomenon of level rearrangement is observed, similar to the Zel'dovich effect, by the onset of an additional stable level which is eventually absorbed by the two-body threshold energy, and can be interpreted as an Efimov-like state of the short-range potential.

  17. Synthetically Useful Base Induced Rearrangements of Aldonolactones

    DEFF Research Database (Denmark)

    Lundt, Inge; Madsen, Robert

    2001-01-01

    Aldonolactones can be activated at the alpha and omega positions by selective bromination or tosylation. The activated aldonolactones can be transformed into epoxyaldonolactones by treatment with base under non-aqueous conditions. Treatment of epoxy- or bromodeoxyaldonolactones with aqueous base...... gives epoxyaldonates in which the epoxide can undergo Payne rearrangement to more stable epoxyaldonates. These can subsequently be opened by the carboxylate group with inversion of the configuration at the attacked carbon. Using this method a number of less available aldonolactones/acids have been...... prepared, in a reaction sequence where the configuration at one, two or three carbon centers has been stereospecifically interconverted. An attractive synthesis Of L-gluconic acid from D-gluconolactone is presented....

  18. Árni Magnússon's rearrangement of paper manuscripts

    DEFF Research Database (Denmark)

    Stegmann, Beeke

    Árni Magnússon’s rearrangement of paper manuscripts draws attention to the early history of Árni Magnússon’s(1663-1730) manuscript collection. The thesis examines Árni’s extensive rearrangement of paper manuscripts, showing that he repeatedly altered the physical composition of codices in his...

  19. Uniparental disomy analysis in carriers of balanced chromosome rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    May, K.M.; Pettay, D.; Muralidharan, K. [Emory Univ. School of Medicine, Atlanta, GA (United States)] [and others

    1994-09-01

    Although most individuals who carry a balanced familial chromosome rearrangement are phenotypically normal, those who are clinically abnormal raise the question of whether or not the rearrangement plays a causative role. One possible mechanism involves meiotic segregation of a normal homolog along with the rearranged chromosome(s) such that a trisomic conception occurs. Subsequent loss by mitotic nondisjunction of the structurally normal chromosome contributed by the non-carrier parent would then result in uniparental disomy (UPD) in a conceptus carrying a balanced rearrangement. UPD for chromosomes 14 and 15 has been demonstrated in several clinically abnormal individuals who carry a familial Robertsonian translocation. We have extended this type of analysis to include other forms of balanced chromosome rearrangements. We report the results of UPD analysis of 14 families who have a phenotypically abnormal child with an apparently balanced rearrangement. The series includes 4 reciprocal translocations, 4 Robertsonian translocations, 2 X;autosome translocations, and 4 inversions. High resolution chromosomes were used to compare breakpoints between parent and offspring to exclude the possibility of further rearrangements. Parental origin of the chromosome(s) involved was determined by DNA polymorphism analysis using PCR or Southern blotting techniques. We found no evidence of UPD in any of the 14 cases. Our data suggest that UPD is not a common explanation for phenotypically abnormal carriers of balanced chromosome rearrangements.

  20. Árni Magnússon's rearrangement of paper manuscripts

    DEFF Research Database (Denmark)

    Stegmann, Beeke

    Árni Magnússon’s rearrangement of paper manuscripts draws attention to the early history of Árni Magnússon’s (1663-1730) manuscript collection. The thesis examines Árni’s extensive rearrangement of paper manuscripts, showing that he repeatedly altered the physical composition of codices in his...

  1. Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL

    Energy Technology Data Exchange (ETDEWEB)

    Do, To Uyen [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Ho, Bay; Shih, Shyh-Jen [Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States); Vaughan, Andrew, E-mail: Andrew.vaughan@ucdmc.ucdavis.edu [Graduate Group in Immunology, University of California Davis, Davis, CA 95616 (United States); Department of Radiation Oncology, University of California Davis, Sacramento CA 95817 (United States)

    2012-12-15

    Highlights: ► A Zinc Finger Nuclease (ZFN) targeting a leukemogenic hot spot for rearrangement in MLL is created. ► The novel ZFN efficiently cleaves MLL exon 13. ► Despite MLL cleavage and evidence of mis-repair, no leukemogenic translocations were produced. ► MLL cleavage alone is insufficient to generate leukemogenic translocations. - Abstract: Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.

  2. Clinicopathological features of a squamous cell carcinoma of the lung harboring ALK rearrangement and with crizotinib responsivity%ALK阳性并克唑替尼治疗有效肺鳞状细胞癌临床病理分析

    Institute of Scientific and Technical Information of China (English)

    穆晶; 吴卫华; 蔡毅然; 苏丹; 张海青

    2015-01-01

    Objective To observe the clinicopathological characteristics of the patient with ALK rearrangement in squamous cell carcinoma of the lung(SCCL)and analyze the responsivity of clinical therapy and prognosis of the patient. To explore the necessity of ALK testing in SCCL. Methods A 54-year-old woman was involved in this observation. The clinical records,computerized tomographic checkup,pathological morphol-ogy and immunohistochemistry of the case were discussed. ALK rearrangement was screened by using immunohistochemistry on Benchmark XT au-tostainer. Results A 54-year-old,female,never-smoking patient presented with left back pain and cough,expectoration for two months. Com-puted tomography revealed a mass in left hilar area with bronchial stenosis in superior lobe of the left lung. Bronchoscopic exploration showed that a mass prominent to the cavity of left upper lobe bronchus and blocked airway. MRI scanning of the brain and bone scanning verified the presence of metastasis. She was diagnosed as squamous cell carcinoma without EGFR mutation. Ventana immunohistochemistry showed positive ALK expression. This case was administrated gemcitabine combined with cisplatin as first-line chemotherapy for 2 cycles,but tumor still got progression;however, when she received crizotinib therapy,the primary tumor shrank. Progression-free survival was 6 months. Conclusion ALK rearrangement in SCCL is uncommon,crizotinib showed effective in ALK-positive SCCL. So it is necessary for SCCL to screen ALK rearrangement.%目的:观察间变性淋巴瘤激酶( ALK)融合基因阳性肺鳞状细胞癌( SCCL)临床病理特征、临床治疗效果,探讨对肺鳞癌患者进行ALK融合基因检测的必要性。方法 Ventana全自动免疫组化染色检测l例ALK蛋白阳性肺鳞状细胞癌患者,结合临床及影像学资料,观察组织学形态和常规免疫组化染色,分析患者临床治疗及预后。结果患者女性,54岁,不吸烟,因左背

  3. Symmetric Rearrangements Around Infinity with Applications to Levy Processes

    CERN Document Server

    Drewitz, Alexander; Sun, Rongfeng

    2011-01-01

    We prove a new rearrangement inequality for multiple integrals, which partly generalizes a result of Friedberg and Luttinger (1976) and can be interpreted as involving symmetric rearrangements of domains around infinity. As applications, we prove two comparison results for general Levy processes and their symmetric rearrangements. The first application concerns the survival probability of a point particle in a Poisson field of moving traps following independent Levy motions. We show that the survival probability can only increase if the point particle does not move, and the traps and the Levy motions are symmetrically rearranged. This essentially generalizes an isoperimetric inequality of Peres and Sousi (2011) for the Wiener sausage. In the second application, we show that the q-capacity of a Borel measurable set for a Levy process can only increase if the set and the Levy process are symmetrically rearranged. This result generalizes an inequality obtained by Watanabe (1983) for symmetric Levy processes.

  4. Micromechanics Analysis Code With Generalized Method of Cells (MAC/GMC): User Guide. Version 3

    Science.gov (United States)

    Arnold, S. M.; Bednarcyk, B. A.; Wilt, T. E.; Trowbridge, D.

    1999-01-01

    The ability to accurately predict the thermomechanical deformation response of advanced composite materials continues to play an important role in the development of these strategic materials. Analytical models that predict the effective behavior of composites are used not only by engineers performing structural analysis of large-scale composite components but also by material scientists in developing new material systems. For an analytical model to fulfill these two distinct functions it must be based on a micromechanics approach which utilizes physically based deformation and life constitutive models and allows one to generate the average (macro) response of a composite material given the properties of the individual constituents and their geometric arrangement. Here the user guide for the recently developed, computationally efficient and comprehensive micromechanics analysis code, MAC, who's predictive capability rests entirely upon the fully analytical generalized method of cells, GMC, micromechanics model is described. MAC/ GMC is a versatile form of research software that "drives" the double or triply periodic micromechanics constitutive models based upon GMC. MAC/GMC enhances the basic capabilities of GMC by providing a modular framework wherein 1) various thermal, mechanical (stress or strain control) and thermomechanical load histories can be imposed, 2) different integration algorithms may be selected, 3) a variety of material constitutive models (both deformation and life) may be utilized and/or implemented, and 4) a variety of fiber architectures (both unidirectional, laminate and woven) may be easily accessed through their corresponding representative volume elements contained within the supplied library of RVEs or input directly by the user, and 5) graphical post processing of the macro and/or micro field quantities is made available.

  5. Ultrasound guided neural stem cell transplantation through the lateral ventricle for treatment of cerebral palsy in children☆

    Institute of Scientific and Technical Information of China (English)

    Sheng He; Zuo Luan; Suqing Qu; Xuan Qiu; Daqing Xin; Wenkai Jia; Yanhua Shen; Zehui Yu; Tao Xu

    2012-01-01

    A total of 24 children with cerebral palsy were enrolled in this study and underwent ultrasound guided transplantation of neural stem cells through the lateral ventricle. Neural stem cells (3.8 × 106–7.3 × 107) were injected into the lateral ventricles. Mild injury of lateral ventricular blood vessels occurred in only two cases (8.3%). Seven cases (29.2%) experienced a fever. Clinical manifestations were improved to varying degrees in eight cases (28.0%) within 3 months after transplantation. Patient condition did not worsen, and no patient experienced severe adverse reactions.

  6. Genome-wide signatures of 'rearrangement hotspots' within segmental duplications in humans.

    Directory of Open Access Journals (Sweden)

    Mohammed Uddin

    Full Text Available The primary objective of this study was to create a genome-wide high resolution map (i.e., >100 bp of 'rearrangement hotspots' which can facilitate the identification of regions capable of mediating de novo deletions or duplications in humans. A hierarchical method was employed to fragment segmental duplications (SDs into multiple smaller SD units. Combining an end space free pairwise alignment algorithm with a 'seed and extend' approach, we have exhaustively searched 409 million alignments to detect complex structural rearrangements within the reference-guided assembly of the NA18507 human genome (18× coverage, including the previously identified novel 4.8 Mb sequence from de novo assembly within this genome. We have identified 1,963 rearrangement hotspots within SDs which encompass 166 genes and display an enrichment of duplicated gene nucleotide variants (DNVs. These regions are correlated with increased non-allelic homologous recombination (NAHR event frequency which presumably represents the origin of copy number variations (CNVs and pathogenic duplications/deletions. Analysis revealed that 20% of the detected hotspots are clustered within the proximal and distal SD breakpoints flanked by the pathogenic deletions/duplications that have been mapped for 24 NAHR-mediated genomic disorders. FISH Validation of selected complex regions revealed 94% concordance with in silico localization of the highly homologous derivatives. Other results from this study indicate that intra-chromosomal recombination is enhanced in genic compared with agenic duplicated regions, and that gene desert regions comprising SDs may represent reservoirs for creation of novel genes. The generation of genome-wide signatures of 'rearrangement hotspots', which likely serve as templates for NAHR, may provide a powerful approach towards understanding the underlying mutational mechanism(s for development of constitutional and acquired diseases.

  7. Poor response to gefitinib in lung adenocarcinoma with concomitant epidermal growth factor receptor mutation and anaplastic lymphoma kinase rearrangement.

    Science.gov (United States)

    Zhou, Jianya; Zheng, Jing; Zhao, Jing; Sheng, Yihong; Ding, Wei; Zhou, Jianying

    2015-03-01

    A patient presenting with concomitant epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation is rare. We report a non-small cell lung cancer (NSCLC) patient with concomitant ALK rearrangement and exon 19 (E746-A750del) EGFR mutation. The ALK rearrangement was confirmed not only in the primary tumor biopsy specimen, but also in the pleural effusion cell block by reverse transcriptase-polymerase chain reaction (RT-PCR), Ventana ALK immunohistochemistry assay, and fluorescence in situ hybridization. No clinical benefit using chemotherapy or EGFR tyrosine kinase inhibitor gefitinib was obtained in this case.

  8. Muller glia, vision-guided ocular growth, retinal stem cells, and a little serendipity: the Cogan lecture.

    Science.gov (United States)

    Fischer, Andy J

    2011-09-29

    Hypothesis-driven science is expected to result in a continuum of studies and findings along a discrete path. By comparison, serendipity can lead to new directions that branch into different paths. Herein, I describe a diverse series of findings that were motivated by hypotheses, but driven by serendipity. I summarize how investigations into vision-guided ocular growth in the chick eye led to the identification of glucagonergic amacrine cells as key regulators of ocular elongation. Studies designed to assess the impact of the ablation of different types of neurons on vision-guided ocular growth led to the finding of numerous proliferating cells within damaged retinas. These proliferating cells were Müller glia-derived retinal progenitors with a capacity to produce new neurons. Studies designed to investigate Müller glia-derived progenitors led to the identification of a domain of neural stem cells that form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Accelerated ocular growth, caused by visual deprivation, stimulated the proliferation of CMZ progenitors. We formulated a hypothesis that growth-regulating glucagonergic cells may regulate both overall eye size (scleral growth) and the growth of the retina (proliferation of CMZ cells). Subsequent studies identified unusual types of glucagonergic neurons with terminals that ramify within the CMZ; these cells use visual cues to control equatorial ocular growth and the proliferation of CMZ cells. Finally, while studying the signaling pathways that stimulate CMZ and Müller glia-derived progenitors, serendipity led to the discovery of a novel type of glial cell that is scattered across the inner retinal layers.

  9. Recurrent rearrangement during adaptive evolution in an interspecific yeast hybrid suggests a model for rapid introgression.

    Directory of Open Access Journals (Sweden)

    Barbara Dunn

    2013-03-01

    Full Text Available Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ~1-kb region of chromosome 14, and all producing an "interspecific fusion junction" within the MEP2 gene coding sequence, such that the 5' portion derives from S. cerevisiae and the 3' portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a

  10. In vivo models for cancer stem cell research: a practical guide for frequently used animal models and available biomarkers.

    Science.gov (United States)

    Skidan, I; Steiniger, S C J

    2014-04-01

    The identification of a rare population of cancer stem cells whose presence in tumors is believed to determine their growth and metastatic activity, has provided a novel approach for targeted anti-cancer therapy. At the in vivo stage of the development of new therapeutic approaches for killing cancer stem cells, the most significant issues are the appropriate choice of rational animal models that offer the option to select animal species, strains and substrains, essential techniques for the inoculation of tumors, and methods of tumor detection in animals. The identification and validation of various types of cancer stem cell markers, which could serve as potential marker(s) of therapeutic efficacy of applied drugs, is a considerable challenge. The aim of this review is to provide a guide for the in vivo study of novel therapeutics that target cancer stem cells. This review describes frequently used mouse solid tumor models and evaluates their usefulness for cancer stem cell research. The classification of existing compounds that are used in today's experimental anti-cancer stem cell therapy and examples of exploratory first-in-human studies using these compounds for selective elimination of cancer stem cells will also be discussed. Finally, this review will examine the current status of available cancer stem cell markers, and highlight several important cancer stem cell properties that are still not well understood, but could influence the anti-cancer drug development process.

  11. Immunoglobulin gene expression and regulation of rearrangement in kappa transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Ritchie, K.A.

    1986-01-01

    Transgenic mice were produced by microinjection of the functionally rearranged immunoglobulin kappa gene from the myeloma MOPC-21 into the male pronucleus of fertilized mouse eggs, and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were detected by hybridizing tail DNA dots with radioactively labelled pBR322 plasmid DNA, which detects pBR322 sequences left as a tag on the microinjected DNA. Mice that integrated the injected gene (six males) were mated and the DNA, RNA and serum kappa chains of their offspring were analyzed. A rabbit anti-mouse kappa chain antiserum was also produced for use in detection of mouse kappa chains on protein blots. Hybridomas were produced from the spleen cells of these kappa transgenic mice to immortalize representative B cells and to investigate expression of the transgenic kappa gene, its effect on allelic exclusion, and its effect on the control of light chain gene rearrangement and expression. The results show that the microinjected DNA is integrated as concatamers in unique single or, rarely, two separate sites in the genome. The concatamers are composed of several copies (16 to 64) of injected DNA arranged in a head to tail fashion. The transgene is expressed into protein normally and in a tissue specific fashion. For the first time in these transgenic mice, all tissues contain a functionally rearranged and potentially expressible immunoglobulin gene. The transgene is expressed only in B cells and not in hepatocytes, for example. This indicates that rearrangement of immunoglobulin genes is necessary but not sufficient for the tissue specific expression of these genes by B cells.

  12. Rapid induction of orthotopic hepatocellular carcinoma in immune-competent rats by non-invasive ultrasound-guided cells implantation

    Directory of Open Access Journals (Sweden)

    Pan Huay-Ben

    2010-07-01

    Full Text Available Abstract Background The fact that prognoses remain poor in patients with advanced hepatocellular carcinoma highlights the demand for suitable animal models to facilitate the development of anti-cancer medications. This study employed a relatively non-invasive approach to establish an orthotopic hepatocellular carcinoma model in immune-competent rats. This was done by ultrasound-guided implantation of cancer cells and the model was used to evaluate the therapeutic efficacy of short-term and low-dose epirubicin chemotherapy. Methods Rat Novikoff hepatoma cells were injected percutaneously into the liver lobes of Sprague-Dawley rats under the guidance of high resolution ultrasound. The implantation rate and the correlation between dissected and ultrasound-measured tumor sizes were evaluated. A similar induction procedure was performed by means of laparotomy in a different group of rats. Pairs of tumor measurement were compared by ultrasound and computerized tomography scan. Rats with a successful establishment of the tumor were divided into the treatment (7-day low-dose epirubicin group and the control group. The tumor sizes were non-invasively monitored by the same ultrasound machine. Blood and tumor tissues from tumor-bearing rats were examined by biochemical and histological analysis respectively. Results Ultrasound-guided implantation of Novikoff hepatoma cells led to the formation of orthotopic hepatocellular carcinoma in 60.4% (55/91 of the Sprague-Dawley rats. Moreover, tumor sizes measured by ultrasound significantly correlated with those measured by calipers after sacrificing the animals (P Conclusions Ultrasound-guided implantation of Novikoff hepatoma cells is an effective means of establishing orthotopic hepatocellular carcinoma in Sprague-Dawley rats. Short-term and low-dose epirubicin chemotherapy had perturbed tumor progression by inducing apoptosis and neovascularization blockade.

  13. Particle-in-Cell Simulations of Collisionless Magnetic Reconnection with a Non-Uniform Guide Field

    CERN Document Server

    Wilson, Fiona; Hesse, Michael; Harrison, Michael G; Stark, Craig R

    2015-01-01

    Results are presented of a first study of collisionless magnetic reconnection starting from a recently found exact nonlinear force-free Vlasov-Maxwell equilibrium. The initial state has a Harris sheet magnetic field profile in one direction and a non-uniform guide field in a second direction, resulting in a spatially constant magnetic field strength as well as a constant initial plasma density and plasma pressure. It is found that the reconnection process initially resembles guide field reconnection, but that a gradual transition to anti-parallel reconnection happens as the system evolves. The time evolution of a number of plasma parameters is investigated, and the results are compared with simulations starting from a Harris sheet equilibrium and a Harris sheet plus constant guide field equilibrium.

  14. Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca(2.).

    Science.gov (United States)

    Waldeck-Weiermair, Markus; Malli, Roland; Parichatikanond, Warisara; Gottschalk, Benjamin; Madreiter-Sokolowski, Corina T; Klec, Christiane; Rost, Rene; Graier, Wolfgang F

    2015-10-22

    Mitochondrial Ca(2+) uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca(2+) uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca(2+) channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca(2+) signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca(2+) rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca(2+) uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca(2+) rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca(2+) concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

  15. T细胞受体γ引物组合检测蕈样肉芽肿基因重排的研究%Application of BIOMED-2 primers in the detection of T cell receptor γ gene rearrangements in patients with mycosis fungoides

    Institute of Scientific and Technical Information of China (English)

    陈柳青; 陈金波; 段逸群; 李东升; 董碧麟; 张红梅; 俞鑫

    2013-01-01

    Objective To estimate the value of BIOMED-2 primers for the detection of T cell receptor γ (TCR-γ) gene rearrangements in different types of specimens from patients with mycosis fungoides (MF).Methods Totally,15 paraffin-embedded tissue specimens,14 fresh tissue specimens and 18 whole blood specimens were obtained from 28 patients with MF,and subjected to DNA extraction.BIOMED-2 multiplex PCR tubes TCRγ (A+B) were used for the analysis of TCRγgene rearrangements.Data were processed by SPSS 13.0 software,and statistical analysis was done by chi-square test and Fisher's exact probability test.Results TCR-γ gene rearrangements were detected in 3 paraffin-embedded tissue specimens,11 fresh tissue specimens and 12 blood specimens,with significant differences in the detection rate between the three samples (x2 =13.047,P < 0.01).The fresh tissue samples showed a significantly higher detection rate than the paraffin-embedded tissue samples (X2 =12.523,P < 0.01).The detection rate of TCRγgene rearrangements was 3/6 in paraffin-embedded tissue samples collected in 2011,significantly higher than that in the other 9 paraffin-embedded tissue samples collected before 2011 (Fisher's exact probability test,P =0.044),but similar to that in 14 fresh tissue specimens (12/14,Fisher's exact probability test,P =0.044).Decreased detection rate of TCRγ gene rearrangements was observed in blood samples compared with fresh tissue specimens,but no statistical difference was observed between the two types of specimens (x2 =2.358,P > 0.05).Conclusions BIOMED-2 multiplex PCR tubes TCRγ(A+B) are suitable for the detection of clonal rearrangements of TCRγgene in different types of specimens,especially in fresh tissue specimens,from patients with MF.%目的 探讨BIOMED-2系统T细胞受体(TCR)γ引物组合在蕈样肉芽肿(MF)患者不同来源标本中TCRγ基因重排检测中的价值.方法 收集28例MF患者15份石蜡组织样本、14份新鲜皮损及18份全血

  16. In vivo integration of poly(ε-caprolactone)/gelatin nanofibrous nerve guide seeded with teeth derived stem cells for peripheral nerve regeneration.

    Science.gov (United States)

    Beigi, Mohammad-Hossein; Ghasemi-Mobarakeh, Laleh; Prabhakaran, Molamma P; Karbalaie, Khadijeh; Azadeh, Hamid; Ramakrishna, Seeram; Baharvand, Hossein; Nasr-Esfahani, Mohammad-Hossein

    2014-12-01

    Artificial nanofiber nerve guides have gained huge interest in bridging nerve gaps and associated peripheral nerve regeneration due to its high surface area, flexibility and porous structure. In this study, electrospun poly (ε-caprolactone)/gelatin (PCL/Gel) nanofibrous mats were fabricated, rolled around a copper wire and fixed by medical grade adhesive to obtain a tubular shaped bio-graft, to bridge 10 mm sciatic nerve gap in in vivo rat models. Stem cells from human exfoliated deciduous tooth (SHED) were transplanted to the site of nerve injury through the nanofibrous nerve guides. In vivo experiments were performed in animal models after creating a sciatic nerve gap, such that the nerve gap was grafted using (i) nanofiber nerve guide (ii) nanofiber nerve guide seeded with SHED (iii) suturing, while an untreated nerve gap remained as the negative control. In vitro cell culture study was carried out for primary investigation of SHED-nanofiber interaction and its viability within the nerve guides after 2 and 16 weeks of implantation time. Walking track analysis, plantar test, electrophysiology and immunohistochemistry were performed to evaluate functional recovery during nerve regeneration. Vascularization was also investigated by hematoxilin/eosine (H&E) staining. Overall results showed that the SHED seeded on nanofibrous nerve guide could survive and promote axonal regeneration in rat sciatic nerves, whereby the biocompatible PCL/Gel nerve guide with cells can support axonal regeneration and could be a promising tissue engineered graft for peripheral nerve regeneration.

  17. Fuel Cell Power Model Version 2: Startup Guide, System Designs, and Case Studies. Modeling Electricity, Heat, and Hydrogen Generation from Fuel Cell-Based Distributed Energy Systems

    Energy Technology Data Exchange (ETDEWEB)

    Steward, D.; Penev, M.; Saur, G.; Becker, W.; Zuboy, J.

    2013-06-01

    This guide helps users get started with the U.S. Department of Energy/National Renewable Energy Laboratory Fuel Cell Power (FCPower) Model Version 2, which is a Microsoft Excel workbook that analyzes the technical and economic aspects of high-temperature fuel cell-based distributed energy systems with the aim of providing consistent, transparent, comparable results. This type of energy system would provide onsite-generated heat and electricity to large end users such as hospitals and office complexes. The hydrogen produced could be used for fueling vehicles or stored for later conversion to electricity.

  18. Percutaneous CT-Guided Cryoablation as an Alternative Treatment for an Extensive Pelvic Bone Giant Cell Tumor

    Energy Technology Data Exchange (ETDEWEB)

    Panizza, Pedro Sergio Brito; Albuquerque Cavalcanti, Conrado Furtado de [Sírio Libânes Hospital, Radiology and Imaged Guided Intervention Service (Brazil); Yamaguchi, Nise Hitomi [Instituto Avanços em Medicina (Brazil); Leite, Claudia Costa; Cerri, Giovanni Guido; Menezes, Marcos Roberto de, E-mail: marcos.menezes@hc.fm.usp.br [Sírio Libânes Hospital, Radiology and Imaged Guided Intervention Service (Brazil)

    2016-02-15

    A giant cell tumor (GCT) is an intermediate grade, locally aggressive neoplasia. Despite advances in surgical and clinical treatments, cases located on the spine and pelvic bones remain a significant challenge. Failure of clinical treatment with denosumab and patient refusal of surgical procedures (hemipelvectomy) led to the use of cryoablation. We report the use of percutaneous CT-guided cryoablation as an alternative treatment, shown to be a minimally invasive, safe, and effective option for a GCT with extensive involvement of the pelvic bones and allowed structural and functional preservation of the involved bones.

  19. Exohedral and skeletal rearrangements in the molecules of fullerene derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Ignat' eva, Daria V; Ioffe, I N; Troyanov, Sergey I; Sidorov, Lev N [Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)

    2011-07-31

    The data on the migration of monoatomic addends, perfluoroalkyl and more complex organic groups in the molecules of fullerene derivatives published mainly in the last decade are analyzed. Skeletal rearrangements of the carbon cage occurring during chemical reactions are considered.

  20. The Petasis-Ferrier rearrangement: developments and applications.

    Science.gov (United States)

    Minbiole, Emily C; Minbiole, Kevin P C

    2016-04-01

    In the mid-1990s, Petasis reexamined a promising but infrequently used rearrangement strategy, the so-called Ferrier-type-II reaction, and provided it with a modern update. Previously, Ferrier had developed a strategy where carbohydrate derivatives would undergo a fragmentation/aldol-type recombination sequence, generating a carbocycle, albeit under the promotion of stoichiometric mercury salts. Petasis' new variant showed the promise to effectively and stereoselectively convert a range of cyclic vinyl acetals to useful tetrahydrofurans and tetrahydropyrans, using less toxic promoters. Since these first reports, the 'Petasis-Ferrier rearrangement' has represented a vibrant area of research and innovation for organic chemists. With numerous applications in complex natural product total synthesis, the utility of the reaction has been resoundingly established. Recent developments have extended the reaction to a broader synthetic context, allowing for in situ generation of rearrangement substrates and more liberal interpretation of what fragmentation/recombination reactions warrant the designation of a Petasis-Ferrier rearrangement.

  1. Rearrangement invariant optimal range for Hardy type operators

    OpenAIRE

    Soria, Javier; Tradacete, Pedro

    2013-01-01

    We characterize, in the context of rearrangement invariant spaces, the optimal range space for a class of monotone operators related to the Hardy operator. The connection between optimal range and optimal domain for these operators is carefully analyzed.

  2. Micropatterned bioimplant with guided neuronal cells to promote tissue reconstruction and improve functional recovery after primary motor cortex insult.

    Science.gov (United States)

    Vaysse, L; Beduer, A; Sol, J C; Vieu, C; Loubinoux, I

    2015-07-01

    With the ever increasing incidence of brain injury, developing new tissue engineering strategies to promote neural tissue regeneration is an enormous challenge. The goal of this study was to design and evaluate an implantable scaffold capable of directing neurite and axonal growth for neuronal brain tissue regeneration. We have previously shown in cell culture conditions that engineered micropatterned PDMS surface with straight microchannels allow directed neurite growth without perturbing cell differentiation and neurite outgrowth. In this study, the micropatterned PDMS device pre-seeded with hNT2 neuronal cells were implanted in rat model of primary motor cortex lesion which induced a strong motor deficit. Functional recovery was assessed by the forelimb grip strength test during 3 months post implantation. Results show a more rapid and efficient motor recovery with the hNT2 neuroimplants associated with an increase of neuronal tissue reconstruction and cell survival. This improvement is also hastened when compared to a direct cell graft of ten times more cells. Histological analyses showed that the implant remained structurally intact and we did not see any evidence of inflammatory reaction. In conclusion, PDMS bioimplants with guided neuronal cells seem to be a promising approach for supporting neural tissue reconstruction after central brain injury.

  3. Kinase Expression and Chromosomal Rearrangements in Papillary Thyroid Cancer Tissues: Investigations at the Molecular and Microscopic Levels

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich; Kwan, Johnson; Lu, Chun-Mei; Ito, Yuko; Wang, Mei; Baumgartner, Adolf; Hayward, Simon W.; Weier, Jingly F.; Zitzelsberger, Horst F.

    2009-07-07

    Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, ret or the neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- or interchromosomal rearrangements have been suggested as a cause of the disease. The 1986 accident at the nuclear power plant in Chernobyl, USSR, led to the uncontrolled release of high levels of radioisotopes. Ten years later, the incidence of childhood papillary thyroid cancer (chPTC) near Chernobyl had risen by two orders of magnitude. Tumors removed from some of these patients showed aberrant expression of the ret RTK gene due to a ret/PTC1 or ret/PTC3 rearrangement involving chromosome 10. However, many cultured chPTC cells show a normal G-banded karyotype and no ret rearrangement. We hypothesize that the 'ret-negative' tumors inappropriately express a different oncogene or have lost function of a tumor suppressor as a result of chromosomal rearrangements, and decided to apply molecular and cytogenetic methods to search for potentially oncogenic chromosomal rearrangements in Chernobyl chPTC cases. Knowledge of the kind of genetic alterations may facilitate the early detection and staging of chPTC as well as provide guidance for therapeutic intervention.

  4. EVI1 is critical for the pathogenesis of a subset of MLL-AF9-rearranged AMLs.

    Science.gov (United States)

    Bindels, Eric M J; Havermans, Marije; Lugthart, Sanne; Erpelinck, Claudia; Wocjtowicz, Elizabeth; Krivtsov, Andrei V; Rombouts, Elwin; Armstrong, Scott A; Taskesen, Erdogan; Haanstra, Jurgen R; Beverloo, H Berna; Döhner, Hartmut; Hudson, Wendy A; Kersey, John H; Delwel, Ruud; Kumar, Ashish R

    2012-06-14

    The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

  5. Analysis of VH gene rearrangement and somatic hypermutation in type 1 autoimmune pancreatitis.

    Science.gov (United States)

    Okumura, Fumihiro; Sakuma, Hidenori; Nakazawa, Takahiro; Hayashi, Kazuki; Naitoh, Itaru; Miyabe, Katsuyuki; Yoshida, Michihiro; Yamashita, Hiroaki; Ohara, Hirotaka; Inagaki, Hiroshi; Joh, Takashi

    2012-05-01

    Type 1 autoimmune pancreatitis (AIP) is the pancreatic manifestation of systemic fibroinflammatory disease called immunoglobulin G4-associated systemic disease. Although this inflammatory process is considered to be a disease with an autoimmune mechanism, its pathogenesis still remains unclear. To clarify the characteristics of B cells infiltrating the lesion, we analyzed the immunoglobulin heavy chain variable region (VH) gene rearrangement and somatic hypermutation of invasive lymphoid cells in type 1 AIP (n= 3), in comparison with obstructive pancreatitis (n= 3) as a control. DNA was extracted from the affected inflammatory lesions. After PCR amplification of the rearranged VH gene, the clones were subcloned, and recombinant clones were randomly selected and sequenced. More than 60 clones per case were analyzed. Monoclonal VH rearrangement was not detected in any of the cases examined. There was no VH family or VH fragment specific to type 1 AIP and obstructive pancreatitis. However, the rate of unmutated VH fragments in type 1 AIP (17%) was higher than that in obstructive pancreatitis (5.1%) (P= 0.010). Our study suggests that an increased rate of unmutated or less mutated VH genes may be characteristic of type 1 AIP and might play a role in the development of this disease.

  6. Gremlin: an interactive visualization model for analyzing genomic rearrangements.

    Science.gov (United States)

    O'Brien, Trevor M; Ritz, Anna M; Raphael, Benjamin J; Laidlaw, David H

    2010-01-01

    In this work we present, apply, and evaluate a novel, interactive visualization model for comparative analysis of structural variants and rearrangements in human and cancer genomes, with emphasis on data integration and uncertainty visualization. To support both global trend analysis and local feature detection, this model enables explorations continuously scaled from the high-level, complete genome perspective, down to the low-level, structural rearrangement view, while preserving global context at all times. We have implemented these techniques in Gremlin, a genomic rearrangement explorer with multi-scale, linked interactions, which we apply to four human cancer genome data sets for evaluation. Using an insight-based evaluation methodology, we compare Gremlin to Circos, the state-of-the-art in genomic rearrangement visualization, through a small user study with computational biologists working in rearrangement analysis. Results from user study evaluations demonstrate that this visualization model enables more total insights, more insights per minute, and more complex insights than the current state-of-the-art for visual analysis and exploration of genome rearrangements.

  7. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    Science.gov (United States)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  8. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    Science.gov (United States)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  9. Collective Classification of Textual Documents by Guided Self-Organization in T-Cell Cross-Regulation Dynamics

    CERN Document Server

    Abi-Haidar, Alaa; 10.1007/s12065-011-0052-5

    2011-01-01

    We present and study an agent-based model of T-Cell cross-regulation in the adaptive immune system, which we apply to binary classification. Our method expands an existing analytical model of T-cell cross-regulation (Carneiro et al. in Immunol Rev 216(1):48-68, 2007) that was used to study the self-organizing dynamics of a single population of T-Cells in interaction with an idealized antigen presenting cell capable of presenting a single antigen. With agent-based modeling we are able to study the self-organizing dynamics of multiple populations of distinct T-cells which interact via antigen presenting cells that present hundreds of distinct antigens. Moreover, we show that such self-organizing dynamics can be guided to produce an effective binary classification of antigens, which is competitive with existing machine learning methods when applied to biomedical text classification. More specifically, here we test our model on a dataset of publicly available full-text biomedical articles provided by the BioCreat...

  10. The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Ming Zhan

    Full Text Available Embryonic stem cells (ESCs are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs, and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

  11. The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

    Science.gov (United States)

    Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

    2012-01-01

    Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

  12. Clonal T cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in the skin and blood of patients with parapsoriasis and early-stage mycosis fungoides.

    Science.gov (United States)

    Klemke, Claus-Detlev; Dippel, Edgar; Dembinski, Antje; Pönitz, Nina; Assaf, Chalid; Hummel, Michael; Stein, Harald; Goerdt, Sergij

    2002-07-01

    Cutaneous T cell lymphoma (CTCL) and reactive T cell skin diseases represent opposite ends of a spectrum of diseases ranging from overtly malignant to persistently benign. Within this spectrum, the parapsoriasis group is not clearly defined regarding malignant potential. In contrast to consistent findings in advanced-stage CTCL, clonality analysis of parapsoriasis has produced conflicting results in previous studies. As T cell receptor gamma-chain polymerase chain reaction GeneScan analysis (TCR-gamma-PCR-GSA) stands out by its sensitivity, its accuracy in size determination of PCR products, its capacity to identify false positives by repeated analysis and its easy applicability, this approach was used to analyse the clonality status of 41 patients with borderline T cell lymphoproliferative skin diseases, including parapsoriasis (n=27) and early-stage mycosis fungoides (MF) (n=14). A monoclonal T cell infiltrate was demonstrated by repeated TCR-gamma-PCR-GSA in lesional skin specimens in 19.2% of parapsoriasis patients and in 66.6% of early-stage MF cases (p=0.013). In peripheral blood, a monoclonal T cell population was found in a similar percentage of parapsoriasis and of early-stage MF patients (26.7% versus 12.5%; p=0.611). A detailed analysis of parapsoriasis subentities, namely small and large plaque parapsoriasis, and parapsoriasis lichenoides, revealed monoclonality in 2(6)/2(5), 3(14)/2(8) and 0(6)/0/(3) of the skin and peripheral blood specimens, respectively. The high detection rate of false positive cases by repeated analysis (20-37.5%) provides a corrected perspective for the high rates of dominant T cell clones found by others in the peripheral blood of such patients. From the results obtained, three major conclusions can be drawn: firstly, CTCL is clearly associated with detection of monoclonality, even in its early stages; secondly, monoclonality is not a prerequisite for potential CTCL precursor entities; and thirdly, recirculating malignant T

  13. Effect of autologous bone marrow-derived cells associated with guided bone regeneration or not in the treatment of peri-implant defects.

    Science.gov (United States)

    Ribeiro, F V; Suaid, F F; Ruiz, K G S; Rodrigues, T L; Carvalho, M D; Nociti, F H; Sallum, E A; Casati, M Z

    2012-01-01

    This study investigated the effect of bone marrow-derived cells associated with guided bone regeneration in the treatment of dehiscence bone defects around dental implants. Iliac-derived bone marrow cells were harvested from dogs and phenotypically characterized with regard to their osteogenic properties. After teeth extraction, three implant sites were drilled, dehiscences created and implants placed. Dehiscences were randomly assigned to: bone marrow-derived cells, bone marrow-derived cells+guided bone regeneration, and control (no treatment). After 3 months, implants with adjacent tissues were processed histologically, bone-to-implant contact, bone fill within the threads, new bone area in a zone lateral to the implant, new bone height, and new bone weight at the bottom of the defect were determined. Phenotypic characterization demonstrated that bone marrow-derived cells presented osteogenic potential. Statistically higher bone fill within the threads was observed in both bone marrow-derived cells+guided bone regeneration bone marrow-derived cell groups compared with the control group (P0.05). For the other parameters (new bone area, bone-to-implant contact, new bone height and new bone weight), only the bone marrow-derived cells+guided bone regeneration group presented higher values compared with the non-treated control (Pregeneration, although the combined approach seems to be relevant, especially to bone formation out of the implant threads.

  14. Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

    Science.gov (United States)

    Feng, Mingye; Chen, James Y; Weissman-Tsukamoto, Rachel; Volkmer, Jens-Peter; Ho, Po Yi; McKenna, Kelly M; Cheshier, Samuel; Zhang, Michael; Guo, Nan; Gip, Phung; Mitra, Siddhartha S; Weissman, Irving L

    2015-02-17

    Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

  15. Cell-block procedure in endoscopic ultrasound-guided-fine-needle-aspiration of gastrointestinal solid neoplastic lesions

    Institute of Scientific and Technical Information of China (English)

    Antonio; Ieni; Valeria; Barresi; Paolo; Todaro; Rosario; Alberto; Caruso; Giovanni; Tuccari

    2015-01-01

    In the present review we have analyzed the clinical applications of endoscopic ultrasound-guided-fineneedle-aspiration(EUS-FNA) and the methodological aspects obtained by cell-block procedure(CBP) in the diagnostic approach to the gastrointestinal neoplastic pathology. CBP showed numerous advantages in comparison to the cytologic routine smears; in particular, better preservation of cell architecture, achievement of routine haematoxylin-eosin staining equivalent to histological slides and possibility to perform immunohistochemistry or molecular analyses represented the most evident reasons to choose this method. Moreover, by this approach, the differential diagnosis of solid gastrointestinal neoplasias may be more easily achieved and the background of contaminant nonneoplastic gastrointestinal avoided. Finally, biological samples collected by EUS-FNA CBP-assisted should be investigated in order to identify and quantify further potential molecular markers.

  16. Characterization of genetic rearrangements in esophageal squamous carcinoma cell lines by a combination of M-FISH and array-CGH: further confirmation of some split genomic regions in primary tumors

    Directory of Open Access Journals (Sweden)

    Hao Jia-Jie

    2012-08-01

    Full Text Available Abstract Background Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC. Methods Using multiplex-fluorescence in situ hybridization (M-FISH and oligo array-based comparative hybridization (array-CGH, we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. Results M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134 of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM (P = 0.004 and 0.022 and advanced stages (P = 0.004 and 0.039. Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026. Conclusions The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

  17. Oriented cell division: new roles in guiding skin wound repair and regeneration.

    Science.gov (United States)

    Yang, Shaowei; Ma, Kui; Geng, Zhijun; Sun, Xiaoyan; Fu, Xiaobing

    2015-11-18

    Tissue morphogenesis depends on precise regulation and timely co-ordination of cell division and also on the control of the direction of cell division. Establishment of polarity division axis, correct alignment of the mitotic spindle, segregation of fate determinants equally or unequally between daughter cells, are essential for the realization of oriented cell division. Furthermore, oriented cell division is regulated by intrinsic cues, extrinsic cues and other cues, such as cell geometry and polarity. However, dysregulation of cell division orientation could lead to abnormal tissue development and function. In the present study, we review recent studies on the molecular mechanism of cell division orientation and explain their new roles in skin repair and regeneration.

  18. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    Science.gov (United States)

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  19. Unusual maternal uniparental isodisomic x chromosome mosaicism with asymmetric y chromosomal rearrangement.

    Science.gov (United States)

    Lee, B Y; Kim, S Y; Park, J Y; Choi, E Y; Kim, D J; Kim, J W; Ryu, H M; Cho, Y H; Park, S Y; Seo, J T

    2014-01-01

    Infertile men with azoospermia commonly have associated microdeletions in the azoospermia factor (AZF) region of the Y chromosome, sex chromosome mosaicism, or sex chromosome rearrangements. In this study, we describe an unusual 46,XX and 45,X mosaicism with a rare Y chromosome rearrangement in a phenotypically normal male patient. The patient's karyotype was 46,XX[50]/45,X[25]/46,X,der(Y)(pter→q11.222::p11.2→pter)[25]. The derivative Y chromosome had a deletion at Yq11.222 and was duplicated at Yp11.2. Two copies of the SRY gene were confirmed by fluorescence in situ hybridization analysis, and complete deletion of the AZFb and AZFc regions was shown by multiplex-PCR for microdeletion analysis. Both X chromosomes of the predominant mosaic cell line (46,XX) were isodisomic and derived from the maternal gamete, as determined by examination of short tandem repeat markers. We postulate that the derivative Y chromosome might have been generated during paternal meiosis or early embryogenesis. Also, we suggest that the very rare mosaicism of isodisomic X chromosomes might be formed during maternal meiosis II or during postzygotic division derived from the 46,X,der(Y)/ 45,X lineage because of the instability of the derivative Y chromosome. To our knowledge, this is the first confirmatory study to verify the origin of a sex chromosome mosaicism with a Y chromosome rearrangement.

  20. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    Energy Technology Data Exchange (ETDEWEB)

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. (Univ. of Florida College of Medicine, Gainsville, FL (United States)); George, J.F. (Univ. of Alabama, Birmingham, AL (United States)); Goodenow, M.M. (Univ. of Florida College of Medicine, Gainsville, FL (United States) Univ. of Alabama, Birmingham, AL (United States))

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  1. T lymphocyte antigen 4-modified dendritic cell therapy for asthmatic mice guided by the CCR7 chemokine receptor.

    Science.gov (United States)

    Chen, Yan; Wang, Yongming; Fu, Zhou

    2014-08-29

    The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.

  2. Chromosomal rearrangement interferes with meiotic X chromosome inactivation.

    Science.gov (United States)

    Homolka, David; Ivanek, Robert; Capkova, Jana; Jansa, Petr; Forejt, Jiri

    2007-10-01

    Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by the incomplete meiotic synapsis of rearranged chromosomes, by their colocalization with the XY body in primary spermatocytes, and by male-limited sterility. Previously, we argued that such X-autosomal associations could interfere with meiotic sex chromosome inactivation. Recently, supporting evidence has reported modifications of histones in rearranged chromosomes by a process called the meiotic silencing of unsynapsed chromatin (MSUC). Here, we report on the transcriptional down-regulation of genes within the unsynapsed region of the rearranged mouse chromosome 17, and on the subsequent disturbance of X chromosome inactivation. The partial transcriptional suppression of genes in the unsynapsed chromatin was most prominent prior to the mid-pachytene stage of primary spermatocytes. Later, during the mid-late pachytene, the rearranged autosomes colocalized with the XY body, and the X chromosome failed to undergo proper transcriptional silencing. Our findings provide direct evidence on the MSUC acting at the mRNA level, and implicate that autosomal asynapsis in meiosis may cause male sterility by interfering with meiotic sex chromosome inactivation.

  3. Visualization of molecular composition and functionality of cancer cells using nanoparticle-augmented ultrasound-guided photoacoustics

    Science.gov (United States)

    Mallidi, Srivalleesha; Kim, Seungsoo; Karpiouk, Andrei; Joshi, Pratixa P.; Sokolov, Konstantin; Emelianov, Stanislav

    2015-01-01

    Assessment of molecular signatures of tumors in addition to their anatomy and morphology is desired for effective diagnostic and therapeutic procedures. Development of in vivo imaging techniques that can identify and monitor molecular composition of tumors remains an important challenge in pre-clinical research and medical practice. Here we present a molecular photoacoustic imaging technique that can visualize the presence and activity of an important cancer biomarker – epidermal growth factor receptor (EGFR), utilizing the effect of plasmon resonance coupling between molecular targeted gold nanoparticles. Specifically, spectral analysis of photoacoustic images revealed profound changes in the optical absorption of systemically delivered EGFR-targeted gold nanospheres due to their molecular interactions with tumor cells overexpressing EGFR. In contrast, no changes in optical properties and, therefore, photoacoustic signal, were observed after systemic delivery of non-targeted gold nanoparticles to the tumors. The results indicate that multi-wavelength photoacoustic imaging augmented with molecularly targeted gold nanoparticles has the ability to monitor molecular specific interactions between nanoparticles and cell-surface receptors, allowing visualization of the presence and functional activity of tumor cells. Furthermore, the approach can be used for other cancer cell-surface receptors such as human epidermal growth factor receptor 2 (HER2). Therefore, ultrasound-guided molecular photoacoustic imaging can potentially aid in tumor diagnosis, selection of customized patient-specific treatment, and monitor the therapeutic progression and outcome in vivo. PMID:25893171

  4. Simple and Rapid In Vivo Generation of Chromosomal Rearrangements using CRISPR/Cas9 Technology

    Directory of Open Access Journals (Sweden)

    Rafael B. Blasco

    2014-11-01

    Full Text Available Generation of genetically engineered mouse models (GEMMs for chromosomal translocations in the endogenous loci by a knockin strategy is lengthy and costly. The CRISPR/Cas9 system provides an innovative and flexible approach for genome engineering of genomic loci in vitro and in vivo. Here, we report the use of the CRISPR/Cas9 system for engineering a specific chromosomal translocation in adult mice in vivo. We designed CRISPR/Cas9 lentiviral vectors to induce cleavage of the murine endogenous Eml4 and Alk loci in order to generate the Eml4-Alk gene rearrangement recurrently found in non-small-cell lung cancers (NSCLCs. Intratracheal or intrapulmonary inoculation of lentiviruses induced Eml4-Alk gene rearrangement in lung cells in vivo. Genomic and mRNA sequencing confirmed the genome editing and the production of the Eml4-Alk fusion transcript. All mice developed Eml4-Alk-rearranged lung tumors 2 months after the inoculation, demonstrating that the CRISPR/Cas9 system is a feasible and simple method for the generation of chromosomal rearrangements in vivo.

  5. The role of APCDD1 in epithelial rearrangement in tooth morphogenesis.

    Science.gov (United States)

    Neupane, Sanjiv; Sohn, Wern-Joo; Gwon, Gi-Jeong; Kim, Ki-Rim; Lee, Sanggyu; An, Chang-Hyeon; Suh, Jo-Young; Shin, Hong-In; Yamamoto, Hitoshi; Cho, Sung-Won; Lee, Youngkyun; Kim, Jae-Young

    2015-10-01

    Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE.

  6. Conditions for predicting quasistationary states by rearrangement formula

    Science.gov (United States)

    Yamaguchi, Yoshiyuki Y.; Ogawa, Shun

    2015-10-01

    Predicting the long-lasting quasistationary state for a given initial state is one of central issues in Hamiltonian systems having long-range interaction. A recently proposed method is based on the Vlasov description and uniformly redistributes the initial distribution along contours of the asymptotic effective Hamiltonian, which is defined by the obtained quasistationary state and is determined self-consistently. The method, to which we refer as the rearrangement formula, was suggested to give precise prediction under limited situations. Restricting initial states consisting of a spatially homogeneous part and small perturbation, we numerically reveal two conditions that the rearrangement formula prefers: One is a no Landau damping condition for the unperturbed homogeneous part, and the other comes from the Casimir invariants. Mechanisms of these conditions are discussed. Clarifying these conditions, we validate to use the rearrangement formula as the response theory for an external field, and we shed light on improving the theory as a nonequilibrium statistical mechanics.

  7. Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements.

    Science.gov (United States)

    Liu, Pengfei; Erez, Ayelet; Nagamani, Sandesh C Sreenath; Dhar, Shweta U; Kołodziejska, Katarzyna E; Dharmadhikari, Avinash V; Cooper, M Lance; Wiszniewska, Joanna; Zhang, Feng; Withers, Marjorie A; Bacino, Carlos A; Campos-Acevedo, Luis Daniel; Delgado, Mauricio R; Freedenberg, Debra; Garnica, Adolfo; Grebe, Theresa A; Hernández-Almaguer, Dolores; Immken, LaDonna; Lalani, Seema R; McLean, Scott D; Northrup, Hope; Scaglia, Fernando; Strathearn, Lane; Trapane, Pamela; Kang, Sung-Hae L; Patel, Ankita; Cheung, Sau Wai; Hastings, P J; Stankiewicz, Paweł; Lupski, James R; Bi, Weimin

    2011-09-16

    Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.

  8. The Combined C—H Functionalization/Cope Rearrangement: Discovery and Applications in Organic Synthesis

    Science.gov (United States)

    Davies, Huw M. L.; Lian, Yajing

    2012-01-01

    Conspectus The development of methods for the stereoselective functionalization of sp3 C–H bonds is a challenging undertaking. This Account describes the scope of the combined C–H functionalization/Cope rearrangement (CHCR), a reaction that occurs between rhodium-stabilized vinylcarbenoids and substrates containing allylic C–H bonds. Computational studies have shown that the CHCR reaction is initiated by a hydride transfer to the carbenoid from an allyl site of the substrate, which is then rapidly followed by C–C bond formation between the developing rhodium-bound allyl anion and the allyl cation. In principle, the reaction can proceed through four distinct orientations of the vinylcarbenoid and the approaching substrate. The early examples of the CHCR reaction were all highly diastereoselective, consistent with a reaction proceeding via a chair transition state with the vinylcarbenoid adopting an s-cis conformation. Recent computational studies have revealed that other transition state orientations are energetically accessible, and these results have guided the development of highly stereoselective CHCR reactions that proceed through a boat transition state with the vinylcarbenoid in an s-cis configuration. The CHCR reaction has broad applications in organic synthesis. In some new protocols, the CHCR reaction acts as a surrogate to some of the classic synthetic strategies in organic chemistry. The CHCR reaction has served as a synthetic equivalent of the Michael reaction, the vinylogous Mukaiyama aldol reaction, the tandem Claisen rearrangement/Cope rearrangement, and the tandem aldol reaction/siloxy-Cope rearrangement. In all of these cases, the products are generated with very high diastereocontrol. With a chiral dirhodium tetracarboxylate catalyst such as Rh2(S-DOSP)4 or Rh2(S-PTAD)4, researchers can achieve very high levels of asymmetric induction. Applications of the CHCR reaction include the effective enantiodifferentiation of racemic

  9. Submillisecond organic synthesis: Outpacing Fries rearrangement through microfluidic rapid mixing.

    Science.gov (United States)

    Kim, Heejin; Min, Kyoung-Ik; Inoue, Keita; Im, Do Jin; Kim, Dong-Pyo; Yoshida, Jun-ichi

    2016-05-01

    In chemical synthesis, rapid intramolecular rearrangements often foil attempts at site-selective bimolecular functionalization. We developed a microfluidic technique that outpaces the very rapid anionic Fries rearrangement to chemoselectively functionalize iodophenyl carbamates at the ortho position. Central to the technique is a chip microreactor of our design, which can deliver a reaction time in the submillisecond range even at cryogenic temperatures. The microreactor was applied to the synthesis of afesal, a bioactive molecule exhibiting anthelmintic activity, to demonstrate its potential for practical synthesis and production.

  10. Ultrafast infrared studies of complex ligand rearrangements in solution

    Energy Technology Data Exchange (ETDEWEB)

    Payne, Christine K. [Univ. of California, Berkeley, CA (United States)

    2003-01-01

    The complete description of a chemical reaction in solution depends upon an understanding of the reactive molecule as well as its interactions with the surrounding solvent molecules. Using ultrafast infrared spectroscopy it is possible to observe both the solute-solvent interactions and the rearrangement steps which determine the overall course of a chemical reaction. The topics addressed in these studies focus on reaction mechanisms which require the rearrangement of complex ligands and the spectroscopic techniques necessary for the determination of these mechanisms. Ligand rearrangement is studied by considering two different reaction mechanisms for which the rearrangement of a complex ligand constitutes the most important step of the reaction. The first system concerns the rearrangement of a cyclopentadienyl ring as the response of an organometallic complex to a loss of electron density. This mechanism, commonly referred to as ''ring slip'', is frequently cited to explain reaction mechanisms. However, the ring slipped intermediate is too short-lived to be observed using conventional methods. Using a combination of ultrafast infrared spectroscopy and electronic structure calculations it has been shown that the intermediate exists, but does not form an eighteen-electron intermediate as suggested by traditional molecular orbital models. The second example examines the initial steps of alkyne polymerization. Group 6 (Cr, Mo, W) pentacarbonyl species are generated photolytically and used to catalyze the polymerization of unsaturated hydrocarbons through a series of coordination and rearrangement steps. Observing this reaction on the femto- to millisecond timescale indicates that the initial coordination of an alkyne solvent molecule to the metal center results in a stable intermediate that does not rearrange to form the polymer precursor. This suggests that polymerization requires the dissociation of additional carbonyl ligands before

  11. Ultrafast infrared studies of complex ligand rearrangements in solution

    Energy Technology Data Exchange (ETDEWEB)

    Payne, Christine K.

    2003-05-31

    The complete description of a chemical reaction in solution depends upon an understanding of the reactive molecule as well as its interactions with the surrounding solvent molecules. Using ultrafast infrared spectroscopy it is possible to observe both the solute-solvent interactions and the rearrangement steps which determine the overall course of a chemical reaction. The topics addressed in these studies focus on reaction mechanisms which require the rearrangement of complex ligands and the spectroscopic techniques necessary for the determination of these mechanisms. Ligand rearrangement is studied by considering two different reaction mechanisms for which the rearrangement of a complex ligand constitutes the most important step of the reaction. The first system concerns the rearrangement of a cyclopentadienyl ring as the response of an organometallic complex to a loss of electron density. This mechanism, commonly referred to as ''ring slip'', is frequently cited to explain reaction mechanisms. However, the ring slipped intermediate is too short-lived to be observed using conventional methods. Using a combination of ultrafast infrared spectroscopy and electronic structure calculations it has been shown that the intermediate exists, but does not form an eighteen-electron intermediate as suggested by traditional molecular orbital models. The second example examines the initial steps of alkyne polymerization. Group 6 (Cr, Mo, W) pentacarbonyl species are generated photolytically and used to catalyze the polymerization of unsaturated hydrocarbons through a series of coordination and rearrangement steps. Observing this reaction on the femto- to millisecond timescale indicates that the initial coordination of an alkyne solvent molecule to the metal center results in a stable intermediate that does not rearrange to form the polymer precursor. This suggests that polymerization requires the dissociation of additional carbonyl ligands before

  12. Rearrangement and evolution of mitochondrial genomes in parrots.

    Science.gov (United States)

    Eberhard, Jessica R; Wright, Timothy F

    2016-01-01

    Mitochondrial genome rearrangements that result in control region duplication have been described for a variety of birds, but the mechanisms leading to their appearance and maintenance remain unclear, and their effect on sequence evolution has not been explored. A recent survey of mitochondrial genomes in the Psittaciformes (parrots) found that control region duplications have arisen independently at least six times across the order. We analyzed complete mitochondrial genome sequences from 20 parrot species, including representatives of each lineage with control region duplications, to document the gene order changes and to examine effects of genome rearrangements on patterns of sequence evolution. The gene order previously reported for Amazona parrots was found for four of the six independently derived genome rearrangements, and a previously undescribed gene order was found in Prioniturus luconensis, representing a fifth clade with rearranged genomes; the gene order resulting from the remaining rearrangement event could not be confirmed. In all rearranged genomes, two copies of the control region are present and are very similar at the sequence level, while duplicates of the other genes involved in the rearrangement show signs of degeneration or have been lost altogether. We compared rates of sequence evolution in genomes with and without control region duplications and did not find a consistent acceleration or deceleration associated with the duplications. This could be due to the fact that most of the genome rearrangement events in parrots are ancient, and additionally, to an effect of body size on evolutionary rate that we found for mitochondrial but not nuclear sequences. Base composition analyses found that relative to other birds, parrots have unusually strong compositional asymmetry (AT- and GC-skew) in their coding sequences, especially at fourfold degenerate sites. Furthermore, we found higher AT skew in species with control region duplications. One

  13. Beckmann Rearrangement of Erythromycin A 9(E)-Oxime

    Institute of Scientific and Technical Information of China (English)

    邓志华; 姚国伟; 欧育湘

    2003-01-01

    9-Deoxo-6-deoxy-6,9-epoxy-9,9a-didehydro-9a-aza-9a-homoerythromycin A (1), 9-deoxo-11-deoxy-9,11-epoxy-9,9a-didehydro-9a-aza-9a-homoerythromycin A (2) and 9a-aza-9a-homoerythromycin cyclic lactam (3) were synthesized by the Beckmann rearrangement of erythromycin A 9(E)-oxime (4). The structures of compounds (1), (2) and (3) have been identified by their spectral data. The reaction mechanism was also discussed. The yield of the Beckmann rearrangement of compounds (4) was better than that reported in literatures.

  14. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  15. The hitchhikers guide to cancer stem cell theory: markers, pathways and therapy.

    Science.gov (United States)

    Fábián, Ákos; Vereb, György; Szöllősi, János

    2013-01-01

    Cancer stem cell (CSC) biology is a rapidly developing field within cancer research. CSCs are postulated to be a unique cell population exclusively capable of infinite self renewal, multilineage differentiation and with ability to evade conventional cytotoxic cancer therapy. These traits distinguish CSCs from their more differentiated counterparts, which possess only limited or no potential for self renewal and tumor initiation. Therefore, CSCs would be the driving motor of malignant growth and therapy resistance. Accordingly, successful cancer treatment would need to eliminate this highly potent group of cells, since even small residual numbers would suffice to recapitulate the disease after therapy. Putative CSCs has been identified in a broad range of human malignancies and several cell surface markers have been associated with their stem cell phenotype. Despite all efforts, a pure CSC population has not been isolated and often in vitro clonogenic and in vivo tumorigenic potential is found in several cell populations with occasionally contradictory surface marker signatures. Here, we give a brief overview of recent advances in CSC theory, including the signaling pathways in CSCs that also appear crucial for stem cells homeostasis in normal tissues. We discuss evidence for the interaction of CSCs with the stromal tumor environment. Finally, we review the emerging potentially effective CSC-targeted treatment strategies and their future role in therapy.

  16. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  17. Diversity of V delta-J delta gene rearrangement in peripheral blood lymphocytes and intrathecal IgG synthesis in multiple sclerosis.

    Science.gov (United States)

    Michałowska-Wender, G; Nowak, J; Losy, J; Januszkiewicz, D; Wender, M

    1999-01-01

    The object of the study is a comparison of intrathecal IgG synthesis and gamma/delta TCR genes rearrangement in multiple sclerosis. The subgroup of 13 cases with intrathecal IgG synthesis and positive oligoclonal bands was compared with 8 cases with IgG index below 0.75 and with undetectable oligoclonal bands. TCR gene rearrangement was studied in peripheral blood lymphocytes by PCR analysis. In majority of cases of the first group the V delta-J delta junctional repertoire was restricted as evidenced by oligoclonal rearrangement. Monoclonal pattern of rearrangement was also established in some cases concerning V delta 1-J delta 1 and V delta 5-J delta 1. In all cases with one exception, demonstrating IgG index < 0.75 and with negative oligoclonal bands in CSF the oligo- or polyclonal pattern of V delta-J delta gene rearrangement was noticed. It is therefore suggested that subset T and B lymphocytes may undergo clonal expansion in MS as evidenced by restricted pattern of V delta-J delta rearrangement and intrathecal oligoclonal IgG synthesis, respectively. Oligoclonal expansion at certain B and T cells may occur due to stimulation by an antigen related to MS pathogen.

  18. Combination of engineered neural cell adhesion molecules and GDF-5 for improved neurite extension in nerve guide concepts.

    Science.gov (United States)

    Niere, Marc; Braun, Bettina; Gass, Rea; Sturany, Sabine; Volkmer, Hansjürgen

    2006-06-01

    Current therapeutical approaches for the treatment of severe lesions in the peripheral nervous system rely on the use of autologous tissue or the body's own Schwann cells. However, these approaches are limited and alternative strategies for peripheral nerve regeneration are required. Here we evaluate combinations of a variety of neuronal regeneration factors including engineered cell adhesion molecules and growth factors in embryonic model neurons to test the possible improvement of artificial nerve guides by cooperative mechanisms. Cell adhesion molecules L1 and neurofascin synergistically promote neurite elongation. The outgrowth promoting properties of both proteins can be combined and further increased within one chimeric protein. Addition of growth and differentiation factor 5 (GDF-5) further enhances neurite outgrowth in a substrate-independent manner. This effect is not due to a protective mode of action of GDF-5 against pro-apoptotic stimuli. Consequently, the study supports the idea that different modes of action of pro-regenerative factors may contribute synergistically to neurite outgrowth and emphasizes the applicability of combinations of proteins specifically involved in development of the nervous system for therapeutical approaches.

  19. Real-time 3D visualization of cellular rearrangements during cardiac valve formation.

    Science.gov (United States)

    Pestel, Jenny; Ramadass, Radhan; Gauvrit, Sebastien; Helker, Christian; Herzog, Wiebke; Stainier, Didier Y R

    2016-06-15

    During cardiac valve development, the single-layered endocardial sheet at the atrioventricular canal (AVC) is remodeled into multilayered immature valve leaflets. Most of our knowledge about this process comes from examining fixed samples that do not allow a real-time appreciation of the intricacies of valve formation. Here, we exploit non-invasive in vivo imaging techniques to identify the dynamic cell behaviors that lead to the formation of the immature valve leaflets. We find that in zebrafish, the valve leaflets consist of two sets of endocardial cells at the luminal and abluminal side, which we refer to as luminal cells (LCs) and abluminal cells (ALCs), respectively. By analyzing cellular rearrangements during valve formation, we observed that the LCs and ALCs originate from the atrium and ventricle, respectively. Furthermore, we utilized Wnt/β-catenin and Notch signaling reporter lines to distinguish between the LCs and ALCs, and also found that cardiac contractility and/or blood flow is necessary for the endocardial expression of these signaling reporters. Thus, our 3D analyses of cardiac valve formation in zebrafish provide fundamental insights into the cellular rearrangements underlying this process.

  20. 13号染色体重排所致严重少弱畸形精子症患者生精细胞减数分裂分析%Meiotic analysis of spermatogenic cells in severe oligoasthenoteratozoospermia with chromosome 13 rearrangement

    Institute of Scientific and Technical Information of China (English)

    崔英霞; 杨晓玉; 夏欣一; 周洋; 陈颖皓; 胡毓安

    2012-01-01

    Objective: To explore the possible mechanisms of spermatogenic arrest in severe oligoasthenoteratozoospermia induced by supernumerary, ring-neocentric 13ql2. 3 → 13q22 chromosome and reciprocal deletion. Methods: We performed a genomiipwide high-density oaCGH analysis for a case of oligoasthenoteratozoospermia with abnormal chromosome 13 to characterize the breakpoints of the chromosome involved or the gene deletion caused by the rearrangement. We also conducted a fluorescence in situ hybridization analysis on the germ cells using probes of 13ql4/13qter to observe the pairing condition of homologous chromosome 13. Results: We identified by oaCGH analysis a microdeletion of 4 consecutive probes ( A_16_P19757882, A_16_P02744617, A_14_ P108858 and A_16_P02744687 at chrl3ql2.3: 27979261 →28039191) with 59.93 kb between the FLT1 and POMP genes, with no annotated genes in the deleted region. The signals of 13ql4 and 13qter were separated from each other in 90% of all the primary sper-matocytes examined, indicating the unpairing of homologous chromosome 13 or synapse failure. Conclusion: Chromosomal rearrange- merit-induced spermatogenesis failure is caused by the impairing of the homologous chromosomes involved in the first meiotic division of germ cells.%目的:探讨13号染色体长臂相互缺失所致严重少弱畸形精子症患者生精阻滞的可能机制. 方法:对1例13号染色体异常的严重少弱畸形精子症患者血液样本进行全基因组的寡核苷酸微阵列比较基因组杂交分析,以确定受累染色体的断裂点或基因缺失.对患者生殖细胞进行多色荧光原位杂交分析,以观察初级精母细胞13号染色体配对的情况. 结果:寡核苷酸微阵列比较基因组杂交技术显示在13q12.3上有连续4个探针的缺失(A_16_P19757882,A16_P02744617,A_14_ P108858和A_16_P02744687),覆盖59.93 kb,位于基因FLT1和POMP之间,没有注释基因存在.初级精母细胞的减数分裂分析显示同源13

  1. Unveiling ubiquitinome rearrangements induced by Salmonella infection.

    Science.gov (United States)

    Bionda, Tihana; Behrends, Christian

    2016-09-01

    Ubiquitination plays a critical role in the activation of host immune responses to infection and serves as a signal for pathogen delivery to phagophores along the xenophagy pathway. We recently performed systematic ubiquitination site profiling of epithelial cells infected with Salmonella Typhimurium. Our findings specifically highlight components of the NFKB, membrane trafficking pathways and RHO GTPase systems as ubiquitination hubs during infection. In addition, a broad spectrum of bacterial effectors and several outer membrane proteins are ubiquitinated in infected cells. This comprehensive resource of ubiquitinome dynamics during Salmonella infection enables further understanding of the complex host-pathogen interplay and may reveal novel targets for the inhibition of Salmonella invasion and inflammation.

  2. Involvement of interstitial telomeric sequences in two new cases of mosaicism for autosomal structural rearrangements.

    Science.gov (United States)

    Lévy, Jonathan; Receveur, Aline; Jedraszak, Guillaume; Chantot-Bastaraud, Sandra; Renaldo, Florence; Gondry, Jean; Andrieux, Joris; Copin, Henri; Siffroi, Jean-Pierre; Portnoï, Marie-France

    2015-02-01

    Mosaicism for an autosomal structural rearrangement that does not involve ring or marker chromosomes is rare. The mechanisms responsible for genome instability have not always been explained. Several studies have shown that interstitial telomeric sequences (ITSs), involved in some mosaic constitutional anomalies, are potent sources of genomic instability. Here we describe two cases of mosaicism for uncommon constitutional autosomal rearrangements, involving ITSs, identified by karyotyping and characterized by FISH and SNP-array analysis. The first patient, a boy with global developmental delay, had a rare type of pure distal 1q inverted duplication (1q32-qter), attached to the end of the short arm of the same chromosome 1, in approximately 35% of his cells. The second patient, a phenotypically normal man, was diagnosed as having mosaic for a balanced non-reciprocal translocation of the distal segment of 7q (7q33qter), onto the terminal region of the short arm of a whole chromosome 12, in approximately 80% of his cells. The remaining 20% of the cells showed an unbalanced state of the translocation, with only the der(7) chromosome. He was ascertained through his malformed fetus carrying a non-mosaic partial monosomy 7q, identified at prenatal diagnosis. We show that pan-telomeric and subtelomeric sequences were observed at the interstitial junction point of the inv dup(1q) and of the der(12)t(7;12), respectively. The present cases and review of the literature suggest that the presence of ITSs at internal sites of the chromosomes may explain mechanisms of the patients's mosaic structural rearrangements.

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

    Directory of Open Access Journals (Sweden)

    Paula Paulo

    2012-07-01

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

  4. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  5. Salvileucalin B, a novel diterpenoid with an unprecedented rearranged neoclerodane skeleton from Salvia leucantha Cav.

    Science.gov (United States)

    Aoyagi, Yutaka; Yamazaki, Akira; Nakatsugawa, Chihiro; Fukaya, Haruhiko; Takeya, Koichi; Kawauchi, Susumu; Izumi, Hiroshi

    2008-10-16

    Salvileucalin B (2), having an unprecedented rearranged neoclerodane skeleton, was isolated from the aerial parts of Salvia leucantha Cav. (Labiatae) along with salvileucalin A (1). The absolute structures were elucidated by spectroscopic analysis, X-ray crystallographic analysis, and vibrational circular dichroism. Compound 2 represents a novel neoclerodane, characterized by a tricyclo[3.2.1.0 (2,7)]octane substructure incorporating the exocyclic C-20 methylene of 1. This molecule exerted cytotoxic activity against A549 and HT-29 cells with IC50 values of 5.23 and 1.88 microg/mL, respectively.

  6. Inhibition of cell division induced by external guide sequences (EGS Technology targeting ftsZ.

    Directory of Open Access Journals (Sweden)

    Carol Davies Sala

    Full Text Available EGS (external guide sequence technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3(pLysS the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.

  7. A comprehensive comparative analysis of the histomorphological features of ALK-rearranged lung adenocarcinoma based on driver oncogene mutations: frequent expression of epithelial-mesenchymal transition markers than other genotype.

    Directory of Open Access Journals (Sweden)

    Hyojin Kim

    Full Text Available Molecular classification of lung cancer correlates well with histomorphological features. However, specific histomorphological features that differentiate anaplastic lymphoma kinase (ALK-rearranged tumors from ALK-negative tumors have not been fully evaluated. Eighty ALK-rearranged and 213 ALK-negative (91 epidermal growth factor receptor-mutated; 29 K-ras-mutated; 93 triple-negative resected lung adenocarcinomas were analyzed for several histomorphological parameters and histological subtype. ALK-rearranged tumors were associated with younger age at presentation, frequent nodal metastasis, and higher stage of disease at diagnosis. ALK-rearranged tumors were more likely to show a solid predominant pattern than ALK-negative tumors (43.8%; 35/80; p<0.001. Unlike ALK-negative tumors, a lepidic predominant pattern was not observed in ALK-rearranged tumors (p<0.001. In multivariate analysis, the most significant morphological features that distinguished ALK-rearranged tumors from ALK-negative tumors were cribriform formation (odds ratio [OR], 3.253; p = 0.028, presence of mucin-containing cells (OR, 4.899; p = 0.008, close relationship to adjacent bronchioles (OR, 5.361; p = 0.001, presence of psammoma bodies (OR, 4.026; p = 0.002, and a solid predominant pattern (OR, 13.685; p = 0.023. ALK-rearranged tumors exhibited invasive histomorphological features, aggressive behavior and frequent expression of epithelial-mesenchymal transition markers (loss of E-cadherin and expression of vimentin compared with other genotype (p = 0.015. Spatial proximity between bronchus and ALK-rearranged tumors and frequent solid histologic subtype with p63 expression may cause diagnostic difficulties to differentiate squamous cell carcinoma in the small biopsy, whereas p40 was rarely expressed in ALK-rearranged adenocarcinoma. Knowledge of these features may improve the diagnostic accuracy and lead to a better understanding of the characteristic behavior of ALK-rearranged

  8. Preparation and characterization of Boron carbide nanoparticles for use as a novel agent in T cell-guided boron neutron capture therapy

    DEFF Research Database (Denmark)

    Mortensen, M. W.; Sørensen, P. G.; Björkdahl, O.;

    2006-01-01

    Boron carbide nanoparticles are proposed as a system for T cell-guided boron neutron capture therapy. Nanoparticles were produced by ball milling in various atmospheres of commercially available boron carbide. The physical and chemical properties of the particles were investigated using...

  9. Multiple subclasses of Purkinje cells in the primate floccular complex provide similar signals to guide learning in the vestibulo-ocular reflex

    Science.gov (United States)

    Raymond, J. L.; Lisberger, S. G.

    1997-01-01

    The neural "learning rules" governing the induction of plasticity in the cerebellum were analyzed by recording the patterns of neural activity in awake, behaving animals during stimuli that induce a form of cerebellum-dependent learning. We recorded the simple- and complex-spike responses of a broad sample of Purkinje cells in the floccular complex during a number of stimulus conditions that induce motor learning in the vestibulo-ocular reflex (VOR). Each subclass of Purkinje cells carried essentially the same information about required changes in the gain of the VOR. The correlation of simple-spike activity in Purkinje cells with activity in vestibular pathways could guide learning during low-frequency but not high-frequency stimuli. Climbing fiber activity could guide learning during all stimuli tested but only if compared with the activity present approximately 100 msec earlier in either vestibular pathways or Purkinje cells.

  10. Mind over Matter. Teacher's Guide.

    Science.gov (United States)

    National Inst. on Drug Abuse (DHHS/PHS), Rockville, MD.

    This teacher's guide aims to develop an understanding among students in grades 5-9 about the biological effects of drug use. The guide provides background information on the anatomy of the brain, nerve cells and neurotransmission, and the effects of drugs on the brain. Drugs described in this guide include marijuana, opiates, inhalants,…

  11. Unveiling ubiquitinome rearrangements induced by Salmonella infection

    Science.gov (United States)

    Bionda, Tihana; Behrends, Christian

    2016-01-01

    ABSTRACT Ubiquitination plays a critical role in the activation of host immune responses to infection and serves as a signal for pathogen delivery to phagophores along the xenophagy pathway. We recently performed systematic ubiquitination site profiling of epithelial cells infected with Salmonella Typhimurium. Our findings specifically highlight components of the NFKB, membrane trafficking pathways and RHO GTPase systems as ubiquitination hubs during infection. In addition, a broad spectrum of bacterial effectors and several outer membrane proteins are ubiquitinated in infected cells. This comprehensive resource of ubiquitinome dynamics during Salmonella infection enables further understanding of the complex host-pathogen interplay and may reveal novel targets for the inhibition of Salmonella invasion and inflammation. PMID:27467224

  12. Beckmann rearrangement of ketoximes to lactams by triphosphazene catalyst.

    Science.gov (United States)

    Hashimoto, Masaharu; Obora, Yasushi; Sakaguchi, Satoshi; Ishii, Yasutaka

    2008-04-01

    Triphosphazene, 1,3,5-triazo-2,4,6-triphosphorine-2,2,4,4,6,6-chloride (TAPC), was found to be an efficient catalyst for the Beckmann rearrangement of cyclohexanone oxime and cyclododecanone oxime to epsilon-caprolactam and laurolactam, which are raw materials of nylon-6 and nylon-12, respectively.

  13. Sequence features contributing to chromosomal rearrangements in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Russell Spencer-Smith

    Full Text Available Through whole genome sequence alignments, breakpoints in chromosomal synteny can be identified and the sequence features associated with these determined. Alignments of the genome sequences of Neisseria gonorrhoeae strain FA1090, N.gonorrhoeae strain NCCP11945, and N. gonorrhoeae strain TCDC-NG08107 reveal chromosomal rearrangements that have occurred. Based on these alignments and dot plot pair-wise comparisons, the overall chromosomal arrangement of strain NCCP11945 and TCDC-NG08107 are very similar, with no large inversions or translocations. The insertion of the Gonococcal Genetic Island in strain NCCP11945 is the most prominent distinguishing feature differentiating these strains. When strain NCCP11945 is compared to strain FA1090, however, 14 breakpoints in chromosomal synteny are identified between these gonococcal strains. The majority of these, 11 of 14, are associated with a prophage, IS elements, or IS-like repeat enclosed elements which appear to have played a role in the rearrangements observed. Additional rearrangements of small regions of the genome are associated with pilin genes. Evidence presented here suggests that the rearrangements of blocks of sequence are mediated by activation of prophage and associated IS elements and reintegration elsewhere in the genome or by homologous recombination between IS-like elements that have generated inversions.

  14. Interfacial re-arrangement in initial microbial adhesion to surfaces

    NARCIS (Netherlands)

    Busscher, Henk J.; Norde, Willem; Sharma, Prashant K.; van der Mei, Henny C.

    2010-01-01

    Upon initial microbial adhesion to a surface multiple events occur that include interfacial re-arrangements in the region between an adhering organism and a surface Application of physico-chemical mechanisms to explain microbial adhesion to surfaces requires better knowledge of the interfacial re ar

  15. Lipschitz Properties in Variable Exponent Problems via Relative Rearrangement

    Institute of Scientific and Technical Information of China (English)

    Jean-Michel RAKOTOSON

    2010-01-01

    The author first studies the Lipschitz properties of the monotone and relative rearrangement mappings in variable exponent Lebesgue spaces completing the result given in[9].This paper is ended by establishing the Lipschitz properties for quasilinear problems with variable exponent when the right-hand side is in some dual spaces of a suitable Sobolev space associated to variable exponent.

  16. Selenium-mediated synthesis of biaryls through rearrangement.

    Science.gov (United States)

    Shahzad, Sohail A; Vivant, Clotilde; Wirth, Thomas

    2010-03-19

    A new cyclization of beta-keto ester substituted stilbene derivatives using selenium electrophiles in the presence of Lewis acids is described. Substituted naphthols are obtained through cyclization and subsequent 1,2-rearrangement of aryl groups under very mild reaction conditions.

  17. The Basel Problem as a Rearrangement of Series

    Science.gov (United States)

    Benko, David; Molokach, John

    2013-01-01

    We give an elementary solution to the famous Basel Problem, originally solved by Euler in 1735. We square the well-known series for arctan(1) due to Leibniz, and use a surprising relation among the re-arranged terms of this squared series.

  18. Somatic structural rearrangements in genetically engineered mouse mammary tumors

    NARCIS (Netherlands)

    Varela, I.; Klijn, C.N.; Stephens, P.J.; Mudie, L.J.; Stebbings, L.; Galappaththige, D.; Van der Gulden, H.; Schut, E.; Klarenbeek, S.; Campbell, P.J.; Wessels, L.F.A.; Stratton, M.R.; Jonkers, J.; Futreal, P.A.; Adams, D.J.

    2010-01-01

    Background: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and B

  19. Guidance of in vitro migration of human mesenchymal stem cells and in vivo guided bone regeneration using aligned electrospun fibers.

    Science.gov (United States)

    Lee, Ji-hye; Lee, Young Jun; Cho, Hyeong-jin; Shin, Heungsoo

    2014-08-01

    Tissue regeneration is a complex process in which numerous chemical and physical signals are coordinated in a specific spatiotemporal pattern. In this study, we tested our hypothesis that cell migration and bone tissue formation can be guided and facilitated by microscale morphological cues presented from a scaffold. We prepared poly(l-lactic acid) (PLLA) electrospun fibers with random and aligned structures and investigated their effect on in vitro migration of human mesenchymal stem cells (hMSCs) and in vivo bone growth using a critical-sized defect model. Using a polydopamine coating on the fibers, we compared the synergistic effects of chemical signals. The adhesion morphology of hMSCs was consistent with the direction of fiber alignment, whereas the proliferation of hMSCs was not affected. The orientation of fibers profoundly affected cell migration, in which hMSCs cultured on aligned fibers migrated 10.46-fold faster along the parallel direction than along the perpendicular direction on polydopamine-coated PLLA nanofibers. We implanted each fiber type into a mouse calvarial defect model for 2 months. The micro-computed tomography (CT) imaging demonstrated that regenerated bone area was the highest when mice were implanted with aligned fibers with polydopamine coating, indicating a positive synergistic effect on bone regeneration. More importantly, scanning electron microscopy microphotographs revealed that the direction of regenerated bone tissue appeared to be consistent with the direction of the implanted fibers, and transmission electron microscopy images showed that the orientation of collagen fibrils appeared to be overlapped along the direction of nanofibers. Taken together, our results demonstrate that the aligned nanofibers can provide spatial guidance for in vitro cell migration as well as in vivo bone regeneration, which may be incorporated as major instructive cues for the stimulation of tissue regeneration.

  20. CT-guided Bipolar and Multipolar Radiofrequency Ablation (RF Ablation) of Renal Cell Carcinoma: Specific Technical Aspects and Clinical Results

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, C. M., E-mail: christof.sommer@med.uni-heidelberg.de [University Hospital Heidelberg, INF 110, Department of Diagnostic and Interventional Radiology (Germany); Lemm, G.; Hohenstein, E. [Minimally Invasive Therapies and Nuclear Medicine, SLK Kliniken Heilbronn GmbH, Clinic for Radiology (Germany); Bellemann, N.; Stampfl, U. [University Hospital Heidelberg, INF 110, Department of Diagnostic and Interventional Radiology (Germany); Goezen, A. S.; Rassweiler, J. [Clinic for Urology, SLK Kliniken Heilbronn GmbH (Germany); Kauczor, H. U.; Radeleff, B. A. [University Hospital Heidelberg, INF 110, Department of Diagnostic and Interventional Radiology (Germany); Pereira, P. L. [Minimally Invasive Therapies and Nuclear Medicine, SLK Kliniken Heilbronn GmbH, Clinic for Radiology (Germany)

    2013-06-15

    Purpose. This study was designed to evaluate the clinical efficacy of CT-guided bipolar and multipolar radiofrequency ablation (RF ablation) of renal cell carcinoma (RCC) and to analyze specific technical aspects between both technologies. Methods. We included 22 consecutive patients (3 women; age 74.2 {+-} 8.6 years) after 28 CT-guided bipolar or multipolar RF ablations of 28 RCCs (diameter 2.5 {+-} 0.8 cm). Procedures were performed with a commercially available RF system (Celon AG Olympus, Berlin, Germany). Technical aspects of RF ablation procedures (ablation mode [bipolar or multipolar], number of applicators and ablation cycles, overall ablation time and deployed energy, and technical success rate) were analyzed. Clinical results (local recurrence-free survival and local tumor control rate, renal function [glomerular filtration rate (GFR)]) and complication rates were evaluated. Results. Bipolar RF ablation was performed in 12 procedures and multipolar RF ablation in 16 procedures (2 applicators in 14 procedures and 3 applicators in 2 procedures). One ablation cycle was performed in 15 procedures and two ablation cycles in 13 procedures. Overall ablation time and deployed energy were 35.0 {+-} 13.6 min and 43.7 {+-} 17.9 kJ. Technical success rate was 100 %. Major and minor complication rates were 4 and 14 %. At an imaging follow-up of 15.2 {+-} 8.8 months, local recurrence-free survival was 14.4 {+-} 8.8 months and local tumor control rate was 93 %. GFR did not deteriorate after RF ablation (50.8 {+-} 16.6 ml/min/1.73 m{sup 2} before RF ablation vs. 47.2 {+-} 11.9 ml/min/1.73 m{sup 2} after RF ablation; not significant). Conclusions. CT-guided bipolar and multipolar RF ablation of RCC has a high rate of clinical success and low complication rates. At short-term follow-up, clinical efficacy is high without deterioration of the renal function.

  1. Near-infrared (NIR) fluorescence imaging of head and neck squamous cell carcinoma for fluorescence-guided surgery (Conference Presentation)

    Science.gov (United States)

    Moore, Lindsay; Warram, Jason M.; de Boer, Esther; Carroll, William R.; Morlandt, Anthony; Withrow, Kirk P.; Rosenthal, Eben L.

    2016-03-01

    During fluorescence-guided surgery, a cancer-specific optical probe is injected and visualized using a compatible device intraoperatively to provide visual contrast between diseased and normal tissues to maximize resection of cancer and minimize the resection of precious adjacent normal tissues. Six patients with squamous cell carcinomas of the head and neck region (oral cavity (n=4) or cutaneous (n=2)) were injected with an EGFR-targeting antibody (Cetuximab) conjugated to a near-infrared (NIR) fluorescent dye (IRDye800) 3, 4, or 7 days prior to surgical resection of the cancer. Each patient's tumor was then imaged using a commercially available, open-field NIR fluorescence imaging device each day prior to surgery, intraoperatively, and post-operatively. The mean fluorescence intensity (MFI) of the tumor was calculated for each specimen at each imaging time point. Adjacent normal tissue served as an internal anatomic control for each patient to establish a patient-matched "background" fluorescence. Resected tissues were also imaged using a closed-field NIR imaging device. Tumor to background ratios (TBRs) were calculated for each patient using both devices. Fluorescence histology was correlated with traditional pathology assessment to verify the specificity of antibody-dye conjugate binding. Peak TBRs using the open-field device ranged from 2.2 to 11.3, with an average TBR of 4.9. Peak TBRs were achieved between days 1 and 4. This study demonstrated that a commercially available NIR imaging device suited for intraoperative and clinical use can successfully be used with a fluorescently-labeled dye to delineate between diseased and normal tissue in this single cohort human study, illuminated the potential for its use in fluoresence-guided surgery.

  2. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

    Science.gov (United States)

    DNA methylation is an epigenetic mechanism central to the development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. We used whole genome bisulfite sequencing, and found that differentiation of mouse colonic intestinal stem cell...

  3. Optically encoded nanoprobes using single walled carbon nanotube as the building scaffold for magnetic field guided cell imaging.

    Science.gov (United States)

    Wang, Hong; Wang, Zhuyuan; Ye, Minglang; Zong, Shenfei; Li, Mingyue; Chen, Peng; Ma, Xueqin; Cui, Yiping

    2014-02-01

    We construct a novel fluorescent, surface enhanced Raman scattering (SERS) encoded and magnetic nanoprobe for live cell imaging. To fabricate this nanoprobe, single walled carbon nanotube (SWNT) is used as the building scaffold while gold nanoparticles (Au NPs), superparamagnetic iron oxide nanoparticles (SPIONs) and quantum dots (QDs) are employed as the building blocks. Here, Au NPs serve as the SERS substrate and QDs act as the fluorescent agent. Au NPs and SPIONs are first adsorbed on the SWNT via electrostatic interactions. Then a silica layer is coated on the SWNT. Finally, QDs are attached on the silica shell. With such a structure, various optical signals can be readily encoded to the nanoprobe simply by using different Raman molecules and QDs with different emission wavelengths. Experimental results show that the as-prepared nanoprobe exhibits well fluorescence and SERS performance. Furthermore, in vitro experiments demonstrate that the nanoprobe can fulfill magnetic field guided fluorescence and SERS dual mode imaging of live cells. As a fascinating optical encoding material and a multifunctional nanoplatform, the presented nanoprobe holds genuine potential in future biosensing applications.

  4. Expression-guided in silico evaluation of candidate cis regulatory codes for Drosophila muscle founder cells.

    Directory of Open Access Journals (Sweden)

    Anthony A Philippakis

    2006-05-01

    Full Text Available While combinatorial models of transcriptional regulation can be inferred for metazoan systems from a priori biological knowledge, validation requires extensive and time-consuming experimental work. Thus, there is a need for computational methods that can evaluate hypothesized cis regulatory codes before the difficult task of experimental verification is undertaken. We have developed a novel computational framework (termed "CodeFinder" that integrates transcription factor binding site and gene expression information to evaluate whether a hypothesized transcriptional regulatory model (TRM; i.e., a set of co-regulating transcription factors is likely to target a given set of co-expressed genes. Our basic approach is to simultaneously predict cis regulatory modules (CRMs associated with a given gene set and quantify the enrichment for combinatorial subsets of transcription factor binding site motifs comprising the hypothesized TRM within these predicted CRMs. As a model system, we have examined a TRM experimentally demonstrated to drive the expression of two genes in a sub-population of cells in the developing Drosophila mesoderm, the somatic muscle founder cells. This TRM was previously hypothesized to be a general mode of regulation for genes expressed in this cell population. In contrast, the present analyses suggest that a modified form of this cis regulatory code applies to only a subset of founder cell genes, those whose gene expression responds to specific genetic perturbations in a similar manner to the gene on which the original model was based. We have confirmed this hypothesis by experimentally discovering six (out of 12 tested new CRMs driving expression in the embryonic mesoderm, four of which drive expression in founder cells.

  5. Human bone marrow and adipose tissue mesenchymal stem cells: a user's guide.

    Science.gov (United States)

    Mosna, Federico; Sensebé, Luc; Krampera, Mauro

    2010-10-01

    Mesenchymal stem cells (MSCs) are adult stem cells that hold great promise in the field of regenerative medicine. They can be isolated from almost any tissue of the body and display, after expansion, very similar properties and minor differences, probably due to their microenvironment of origin. Expansion in vitro can be obtained in cytokine-free, serum-enriched media, as well as in serum-free, basic fibroblast growth factor-enriched media. A detailed immunophenotypic analysis is required to test the purity of the preparation, but no unique distinguishing marker has been described as yet. Functional assays, that is, differentiation studies in vitro, are needed to prove multilineage differentiation of expanded cells, and demonstration of pluripotency is necessary to identify most immature precursors. MSCs show powerful immunomodulative properties toward most of the cells of the immune system: this strengthens the theoretical rationale for their use also in an allogeneic setting across the major histocompatibility complex (MHC) immunological barriers. Systemic intravenous injection and local use have been tried: after systemic injection, MSCs show a high degree of chemotaxis based on pro-inflammatory cytokines, and localize at inflamed and neoplastic tissues; local regeneration has been improved using synthetic, as well as organic scaffolds. On the other hand, inadequate heterotopic in vivo differentiation and neoplastic transformation are potential risks of this form of cell therapy, even if evidence of this sort has been collected only from studies in mice, and generally after prolonged in vitro expansion. This review tries to provide a detailed technical overview of the methods used for human bone-marrow (BM)-derived and adipose-tissue (AT)-derived MSC isolation, in vitro expansion, and characterization for tissue repair. We chose to use BM-MSCs as a model to describe techniques that have been used for MSC isolation and expansion from very different sources, and

  6. A Cell Biologist’s Field Guide to Aurora Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Christian eDe Groot

    2015-12-01

    Full Text Available Aurora kinases are essential for cell division and are frequently misregulated in human cancers. Based on their potential as cancer therapeutics, a plethora of small molecule Aurora kinase inhibitors have been developed, with a subset having been adopted as tools in cell biology. Here, we fill a gap in the characterization of Aurora kinase inhibitors by using biochemical and cell-based assays to systematically profile a panel of 10 commercially available compounds with reported selectivity for Aurora A (MLN8054, MLN8237, MK-5108, MK-8745, Genentech Aurora Inhibitor 1, Aurora B (Hesperadin, ZM447439, AZD1152-HQPA, GSK1070916 or Aurora A/B (VX-680. We quantify the in vitro effect of each inhibitor on the activity of Aurora A alone, as well as Aurora A and Aurora B bound to fragments of their activators, TPX2 and INCENP, respectively. We also report kinome profiling results for a subset of these compounds to highlight potential off-target effects. In a cellular context, we demonstrate that immunofluorescence-based detection of LATS2 and histone H3 phospho-epitopes provides a facile and reliable means to assess potency and specificity of Aurora A versus Aurora B inhibition, and that G2 duration measured in a live imaging assay is a specific readout of Aurora A activity. Our analysis also highlights variation between HeLa, U2OS and hTERT-RPE1 cells that impacts selective Aurora A inhibition. For Aurora B, all 4 tested compounds exhibit excellent selectivity and do not significantly inhibit Aurora A at effective doses. For Aurora A, MK-5108 and MK-8745 are significantly more selective than the commonly used inhibitors MLN8054 and MLN8237. A crystal structure of an Aurora A/MK-5108 complex that we determined suggests the chemical basis for this higher specificity. Taken together, our quantitative biochemical and cell-based analyses indicate that AZD1152-HQPA and MK-8745 are the best current tools for selectively inhibiting Aurora B and Aurora A

  7. Ligand flexibility and framework rearrangement in a new family of porous metal-organic frameworks

    DEFF Research Database (Denmark)

    Hawxwell, Samuel M; Espallargas, Guillermo Mínguez; Bradshaw, Darren;

    2007-01-01

    Ligand flexibility permits framework rearrangement upon evacuation and gas uptake in a new family of porous MOFs.......Ligand flexibility permits framework rearrangement upon evacuation and gas uptake in a new family of porous MOFs....

  8. Vismodegib: a guide to its use in locally advanced or metastatic basal cell carcinoma.

    Science.gov (United States)

    Lyseng-Williamson, Katherine A; Keating, Gillian M

    2013-02-01

    Vismodegib is the first Hedgehog pathway inhibitor to be approved in the USA, where it is indicated for the treatment of adults with metastatic basal cell carcinoma (BCC), or with locally advanced BCC that has recurred following surgery or who are not candidates for surgery, and who are not candidates for radiation. In an ongoing, noncomparative, phase II trial, oral vismodegib was effective in and had an acceptable tolerability profile in the treatment of patients with locally advanced or metastatic BCC.

  9. Modeling of Shear-Induced Red Blood Cell Migration for Guiding Microfluidic Device Design

    Science.gov (United States)

    Durant, Eden; Higgins, Adam; Sharp, Kendra

    2014-11-01

    Through refinement and extension of a two-phase flow model previously reported for modeling blood in cylindrical flows (Gidaspow, 2009), we have developed a computational model for blood flow in complex microfluidic. Treating plasma as a Newtonian fluid and the Red Blood Cells (RBCs) as a granular phase, whose local concentrations are determined statistically, we have captured the migration of RBCs and concomitant formation of a cell free plasma layer at the channel walls. This model provides us with a three-dimensional distribution of RBCs and the development of the stead-state flow profile, and enables us to study the influence of complex microfluidic geometries, including flow obstacles and varying channel dimensions, on the rate and extent of RBC margination. Simulations on 50 and 100 micron square channels match observed trends including decreasing RBC margination rate in larger channels, increasing RBC margination rate with higher hematocrit, and decreasing cell free layer width with increasing hematocrit. This predictive capability will allow microfluidic devices to be tailored and optimized for specific biomedical applications such as separation of blood constituents.

  10. Dermatoscopy-guided therapy of pigmented basal cell carcinoma with imiquimod*

    Science.gov (United States)

    Husein-ElAhmed, Husein; Fernandez-Pugnaire, Maria Antonia

    2016-01-01

    BACKGROUND Dermatoscopy is a non-invasive diagnostic tool used to examine skin lesions with an optical magnification. It has been suggested as a useful tool for monitoring therapeutic response in lentigo maligna patients treated with imiquimod. OBJECTIVE To examine the accuracy of dermatoscopy as a tool to monitor the therapeutic response of pigmented basal cell carcinoma treated with imiquimod. METHOD The authors designed a prospective study. Patients with pigmented basal cell carcinoma were included and data regarding the dermatoscopy features were collected following the Menzies criteria, prior to initiating the imiquimod treatment. Subsequent dermatoscopic evaluations were performed at weeks 4 and 8, following imiquimod discontinuation. RESULTS Twenty lesions were included. The most common pigmented dermatoscopy features were large blue-grey ovoid nests (80%), followed by blue-grey globules (50%) and leaf-like areas (30%). No spoke wheel areas were observed. In 17 out of 20 patients, a response was noted during the first evaluation at 4 weeks, while the clearance was noted at the second check-up after 8 weeks. In two patients, the clearance was found at the initial evaluation at 4 weeks, while in one patient, the response remained unchanged. Blue-grey globules were the fastest to exhibit clearance (50% at week 4), followed by leaf-like areas (15%) and large blue-grey ovoid nests (6.25%). CONCLUSION According to our results, dermatoscopic evaluation enhances the accuracy in the assessment of the clinical response to imiquimod in pigmented basal cell carcinoma. PMID:28099598

  11. Frequent lambda light chain gene rearrangement and expression in a Ly-1 B lymphoma with a productive kappa chain allele.

    OpenAIRE

    Hardy, R R; Dangl, J L; Hayakawa, K.; Jager, G.; Herzenberg, L.A.

    1986-01-01

    We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continu...

  12. Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    J.F. Mata

    2006-11-01

    Full Text Available Infant acute lymphoblastic leukemia (IALL is characterized by mixed lineage leukemia (MLL gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of £0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively. Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

  13. EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

    Science.gov (United States)

    Bindels, Eric M. J.; Havermans, Marije; Lugthart, Sanne; Erpelinck, Claudia; Wocjtowicz, Elizabeth; Krivtsov, Andrei V.; Rombouts, Elwin; Armstrong, Scott A.; Taskesen, Erdogan; Haanstra, Jurgen R.; Beverloo, H. Berna; Döhner, Hartmut; Hudson, Wendy A.; Kersey, John H.; Kumar, Ashish R.

    2012-01-01

    The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)–rearranged leukemias are EVI1pos. High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1pos MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1neg MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1pos HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9–transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1neg. Moreover, shRNA-mediated knockdown of Evi1 in an Evi1pos MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1pos MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs. PMID:22553314

  14. A hitchhiker's guide to myeloid cell subsets: practical implementation of a novel mononuclear phagocyte classification system

    Directory of Open Access Journals (Sweden)

    Martin eGuilliams

    2015-08-01

    Full Text Available The classification of mononuclear phagocytes as either dendritic cells or macrophages has been mainly based on morphology, the expression of surface markers and assumed functional specialization. We have recently proposed a novel classification system of mononuclear phagocytes based on their ontogeny. Here we discuss the practical application of such a classification system through a number of prototypical examples we have encountered while hitchhiking from one subset to another, across species and between steady state and inflammatory settings. Finally, we discuss the advantages and drawbacks of such a classification system and propose a number of improvements to move from theoretical concepts to concrete guidelines.

  15. STUDYING CONFORMATIONAL REARRANGEMENTS OF PROTEIN WHEN DRYING

    Directory of Open Access Journals (Sweden)

    Danilchenko A. S.

    2016-09-01

    Full Text Available The article gives a review and the experimental data on the nutritional value of eggs protein comprising protein, carbohydrates, minerals and amino acids. Metabolism, the structure and function of each cell, as well as external and internal protective functions are defined by proteins. We consider the study of functions of trace elements found in significant numbers in the eggs, which contribute to health: vitamin D, vitamin B12, choline, folic acid, selenium, lutein and zeaxanthin. The high content of egg protein contributes to greater satiety, weight loss and eye health. We present experimental data on the amino acid composition of the protein and experiments of drying a cooked egg white, placed in a Petri dish. After 10 minutes of drying, the weight of the protein did not change and amounted to 0.1 g, which is 16% of the weight of the original white protein. The photo shows that the protein is lost during drying white and became transparent, and protein grains look like "melted" and partially connected with each other. The data obtained can be used for understanding the operation of the biological process of protein in vivo

  16. Enhancement of microhomology-mediated genomic rearrangements by transient loss of mouse Bloom syndrome helicase.

    Science.gov (United States)

    Yamanishi, Ayako; Yusa, Kosuke; Horie, Kyoji; Tokunaga, Masahiro; Kusano, Kohji; Kokubu, Chikara; Takeda, Junji

    2013-09-01

    Bloom syndrome, an autosomal recessive disorder of the BLM gene, confers predisposition to a broad spectrum of early-onset cancers in multiple tissue types. Loss of genomic integrity is a primary hallmark of such human malignancies, but many studies using disease-affected specimens are limited in that they are retrospective and devoid of an appropriate experimental control. To overcome this, we devised an experimental system to recapitulate the early molecular events in genetically engineered mouse embryonic stem cells, in which cells undergoing loss of heterozygosity (LOH) can be enriched after inducible down-regulation of Blm expression, with or without site-directed DNA double-strand break (DSB) induction. Transient loss of BLM increased the rate of LOH, whose breakpoints were distributed along the chromosome. Combined with site-directed DSB induction, loss of BLM synergistically increased the rate of LOH and concentrated the breakpoints around the targeted chromosomal region. We characterized the LOH events using specifically tailored genomic tools, such as high-resolution array comparative genomic hybridization and high-density single nucleotide polymorphism genotyping, revealing that the combination of BLM suppression and DSB induction enhanced genomic rearrangements, including deletions and insertions, whose breakpoints were clustered in genomic inverted repeats and associated with junctional microhomologies. Our experimental approach successfully uncovered the detailed molecular mechanisms of as-yet-uncharacterized loss of heterozygosities and reveals the significant contribution of microhomology-mediated genomic rearrangements, which could be widely applicable to the early steps of cancer formation in general.

  17. [Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia with P53 deletion and TCR-delta rearrangement in a case].

    Science.gov (United States)

    Xu, Xiaofeng; Yang, Wei; Zhang, Xuejin

    2015-10-01

    OBJECTIVE To study the morphology, immunology, cyto- and molecular genetics of a patient with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), deletion of P53 gene and rearrangement of clonal T cell receptors-delta (TCR-delta) gene. METHODS The cell morphology and immunocytochemistry were analyzed by bone marrow testing and biopsy. Cellular immunology was analyzed by flow cytometry. Genetic analysis was carried out by chromosome karyotyping, fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). Immunoglobulin M (IgM) in serum and urine was assayed by immunofixation electrophoresis. And the effect of chlorambucil therapy was evaluated. RESULTS Bone marrow biopsy suggested that the patient was of B lymphocyte type and had abnormal increase of lymphocytoid plasma cells, which were CD38 and CD138 positive. The patient had a normal male karyotype. FISH and PCR analysis of peripheral blood samples suggested deletion of P53 gene and rearrangement of TCR-delta gene. Immunofixation electrophoresis has detected IgM-kappa in both serum and urine. The patient showed partial response to chlorambucil. CONCLUSION In addition to typical clinical features, bone marrow examination, flow cytometry, histochemistry and immunophenotyping, testing for P53 gene deletion and lymphocyte gene rearrangement can facilitate the diagnosis and treatment of LPL/WM.

  18. Preparation and characterization of Boron carbide nanoparticles for use as a novel agent in T cell-guided boron neutron capture therapy.

    Science.gov (United States)

    Mortensen, M W; Sørensen, P G; Björkdahl, O; Jensen, M R; Gundersen, H J G; Bjørnholm, T

    2006-03-01

    Boron carbide nanoparticles are proposed as a system for T cell-guided boron neutron capture therapy. Nanoparticles were produced by ball milling in various atmospheres of commercially available boron carbide. The physical and chemical properties of the particles were investigated using transmission electron microscopy, photon correlation spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, vibrational spectroscopy, gel electrophoresis and chemical assays and reveal profound changes in surface chemistry and structural characteristics. In vitro thermal neutron irradiation of B16 melanoma cells incubated with sub-100 nm nanoparticles (381.5 microg/g (10)B) induces complete cell death. The nanoparticles alone induce no toxicity.

  19. Implementation of a 3D version of ponderomotive guiding center solver in particle-in-cell code OSIRIS

    Science.gov (United States)

    Helm, Anton; Vieira, Jorge; Silva, Luis; Fonseca, Ricardo

    2016-10-01

    Laser-driven accelerators gained an increased attention over the past decades. Typical modeling techniques for laser wakefield acceleration (LWFA) are based on particle-in-cell (PIC) simulations. PIC simulations, however, are very computationally expensive due to the disparity of the relevant scales ranging from the laser wavelength, in the micrometer range, to the acceleration length, currently beyond the ten centimeter range. To minimize the gap between these despair scales the ponderomotive guiding center (PGC) algorithm is a promising approach. By describing the evolution of the laser pulse envelope separately, only the scales larger than the plasma wavelength are required to be resolved in the PGC algorithm, leading to speedups in several orders of magnitude. Previous work was limited to two dimensions. Here we present the implementation of the 3D version of a PGC solver into the massively parallel, fully relativistic PIC code OSIRIS. We extended the solver to include periodic boundary conditions and parallelization in all spatial dimensions. We present benchmarks for distributed and shared memory parallelization. We also discuss the stability of the PGC solver.

  20. Familial complex chromosomal rearrangement resulting in a recombinant chromosome.

    Science.gov (United States)

    Berend, Sue Ann; Bodamer, Olaf A F; Shapira, Stuart K; Shaffer, Lisa G; Bacino, Carlos A

    2002-05-15

    Familial complex chromosomal rearrangements (CCRs) are rare and tend to involve fewer breakpoints and fewer chromosomes than CCRs that are de novo in origin. We report on a CCR identified in a child with congenital heart disease and dysmorphic features. Initially, the child's karyotype was thought to involve a straightforward three-way translocation between chromosomes 3, 8, and 16. However, after analyzing the mother's chromosomes, the mother was found to have a more complex rearrangement that resulted in a recombinant chromosome in the child. The mother's karyotype included an inverted chromosome 2 and multiple translocations involving chromosomes 3, 5, 8, and 16. No evidence of deletion or duplication that could account for the clinical findings in the child was identified.

  1. Chromosome-specific staining to detect genetic rearrangements

    Science.gov (United States)

    Gray, Joe W.; Pinkel, Daniel; Tkachuk, Douglas; Westbrook, Carol

    2013-04-09

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyzes. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  2. Recent applications of ring-rearrangement metathesis in organic synthesis

    Directory of Open Access Journals (Sweden)

    Sambasivarao Kotha

    2015-10-01

    Full Text Available Ring-rearrangement metathesis (RRM involves multiple metathesis processes such as ring-opening metathesis (ROM/ring-closing metathesis (RCM in a one-pot operation to generate complex targets. RRM delivers complex frameworks that are difficult to assemble by conventional methods. The noteworthy point about this type of protocol is multi-bond formation and it is an atom economic process. In this review, we have covered literature that appeared during the last seven years (2008–2014.

  3. First Claisen Rearrangement Reaction in Ionic Liquids with Microwave Heating

    Institute of Scientific and Technical Information of China (English)

    XU Li-Wen; LI Fu-Wei; XIA Chun-Gu

    2003-01-01

    @@ We have demonstrated the first use of the common ionic liquids, [1] bmimBr, bmimBF4 and bmimPF6 as an environmentally benign solvent for the simple Claisen rearrangement under microwave irradiation. In many cases, the re action was carried out in toxic solvents of high boiling point. [2] Here we reported the first example of Claisen rear rangement reaction in green solvents, ionic liquids, under microwave irradiation.

  4. Fries Rearrangement of Phenyl Acetate over Solid Acid Catalyst

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A silica-supported zirconium based solid acid (ZS) has been used as catalyst for the Fries rearrangement of phenyl acetate (PA). The catalyst showed a higher PA conversion activity and a much higher selectivity for o-hydroxyacetophenone (o-HAP) than for strongly acidic zeolite catalysts. The supported catalyst was characterized by XRD, IR, XPS, pyridine-TPD and the surface area measurements. The catalytic properties were influenced significantly by pretreatment temperature.

  5. Fries Rearrangement of Phenyl Acetate over Solid Acid Catalyst

    Institute of Scientific and Technical Information of China (English)

    CanXiongGUO; YanLIU; 等

    2002-01-01

    A silica-supported zirconium based solid acid (ZS) has been used as catalyst for the Fries rearrangement of phenyl acetate (PA). The catalyst showed a higher PA conversion activity and a much higher selectivity for o-hydroxyacetophenone (o-HAP) than for strongly acidic zeolite catalysts. The supported catalyst was characterized by XRD,IR,XPS,pyridine-TPD and the surface area measurements. The catalytic properties were influenced significantly by pretreatment temperature.

  6. Standard guide for mechanical drive systems for remote operation in hot cell facilities

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 Intent: 1.1.1 The intent of this standard is to provide general guidelines for the design, selection, quality assurance, installation, operation, and maintenance of mechanical drive systems used in remote hot cell environments. The term mechanical drive systems used herein, encompasses all individual components used for imparting motion to equipment systems, subsystems, assemblies, and other components. It also includes complete positioning systems and individual units that provide motive power and any position indicators necessary to monitor the motion. 1.2 Applicability: 1.2.1 This standard is intended to be applicable to equipment used under one or more of the following conditions: 1.2.1.1 The materials handled or processed constitute a significant radiation hazard to man or to the environment. 1.2.1.2 The equipment will generally be used over a long-term life cycle (for example, in excess of two years), but equipment intended for use over a shorter life cycle is not excluded. 1.2.1.3 The ...

  7. Extended Rearrangement Inequalities and Applications to Some Quantitative Stability Results

    Science.gov (United States)

    Lemou, Mohammed

    2016-09-01

    In this paper, we prove a new functional inequality of Hardy-Littlewood type for generalized rearrangements of functions. We then show how this inequality provides quantitative stability results of steady states to evolution systems that essentially preserve the rearrangements and some suitable energy functional, under minimal regularity assumptions on the perturbations. In particular, this inequality yields a quantitative stability result of a large class of steady state solutions to the Vlasov-Poisson systems, and more precisely we derive a quantitative control of the L 1 norm of the perturbation by the relative Hamiltonian (the energy functional) and rearrangements. A general non linear stability result has been obtained by Lemou et al. (Invent Math 187:145-194, 2012) in the gravitational context, however the proof relied in a crucial way on compactness arguments which by construction provides no quantitative control of the perturbation. Our functional inequality is also applied to the context of 2D-Euler systems and also provides quantitative stability results of a large class of steady-states to this system in a natural energy space.

  8. Metalla-cope rearrangements: bridging organic and inorganic chemistry.

    Science.gov (United States)

    Greer, Edyta M; Hoffmann, Roald

    2010-08-26

    Density functional theory calculations are performed to explore both concerted chairlike and boatlike as well as stepwise mechanisms of the Cope rearrangement of two hypothetical metalladienes. An osma-1,5-hexadiene is designed by substituting CH(2) in 1,5-hexadiene by its isolobal analogue, 16-electron Os(PH(3))(4). The energy of activation corresponding to the rearrangement of osma-1,5-hexadiene involving the chairlike saddle point is computed as 37.4 kcal/mol, 3.9 kcal/mol above the energy barrier of the parent 1,5-hexadiene calculated with the same method and basis set, and is 4.5 kcal/mol below that of the boatlike pathway. In another isolobal replacement, the CH in 1,5-hexadiene is substituted by a 15-electron Re(PH(3))(3) fragment. Now the chairlike rearrangement of the rhenia-1,5-hexadiene has an E(a) value of 23.0 kcal/mol, 10.8 kcal/mol less than the energy barrier of the parent 1,5-hexadiene calculated at the same level of theory. The ring inversion of the chair and osma-chair diradical intermediates of the stepwise reaction pathway is also examined and is found in both cases to proceed through a very flat potential energy surface involving twist intermediates.

  9. Extended Rearrangement Inequalities and Applications to Some Quantitative Stability Results

    Science.gov (United States)

    Lemou, Mohammed

    2016-12-01

    In this paper, we prove a new functional inequality of Hardy-Littlewood type for generalized rearrangements of functions. We then show how this inequality provides quantitative stability results of steady states to evolution systems that essentially preserve the rearrangements and some suitable energy functional, under minimal regularity assumptions on the perturbations. In particular, this inequality yields a quantitative stability result of a large class of steady state solutions to the Vlasov-Poisson systems, and more precisely we derive a quantitative control of the L 1 norm of the perturbation by the relative Hamiltonian (the energy functional) and rearrangements. A general non linear stability result has been obtained by Lemou et al. (Invent Math 187:145-194, 2012) in the gravitational context, however the proof relied in a crucial way on compactness arguments which by construction provides no quantitative control of the perturbation. Our functional inequality is also applied to the context of 2D-Euler systems and also provides quantitative stability results of a large class of steady-states to this system in a natural energy space.

  10. Highly variable rates of genome rearrangements between hemiascomycetous yeast lineages.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available Hemiascomycete yeasts cover an evolutionary span comparable to that of the entire phylum of chordates. Since this group currently contains the largest number of complete genome sequences it presents unique opportunities to understand the evolution of genome organization in eukaryotes. We inferred rates of genome instability on all branches of a phylogenetic tree for 11 species and calculated species-specific rates of genome rearrangements. We characterized all inversion events that occurred within synteny blocks between six representatives of the different lineages. We show that the rates of macro- and microrearrangements of gene order are correlated within individual lineages but are highly variable across different lineages. The most unstable genomes correspond to the pathogenic yeasts Candida albicans and Candida glabrata. Chromosomal maps have been intensively shuffled by numerous interchromosomal rearrangements, even between species that have retained a very high physical fraction of their genomes within small synteny blocks. Despite this intensive reshuffling of gene positions, essential genes, which cluster in low recombination regions in the genome of Saccharomyces cerevisiae, tend to remain syntenic during evolution. This work reveals that the high plasticity of eukaryotic genomes results from rearrangement rates that vary between lineages but also at different evolutionary times of a given lineage.

  11. CT-Guided Radiofrequency Ablation of T1a Renal Cell Carcinoma in Korea: Mid-Term Outcomes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hae Jin; Park, Byung Kwan; Park, Jung Jae; Kim, Chan Kyo [Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351 (Korea, Republic of)

    2016-11-01

    To evaluate the mid-term outcomes of percutaneous radiofrequency ablation (RFA) treatment in patients with small (< 4 cm) renal cell carcinoma (RCC) in Korea. Between 2010 and 2015, 51 patients (40 men and 11 women; median age, 57 years) with biopsyproven 51 RCC were treated using CT-guided RFA. All patients were clinically staged T1aN0M0 prior to RFA. The median tumor size and follow-up period were 2.1 cm (range, 1.0–3.9 cm) and 26 months (4–60 months), respectively. Local tumor progression, distant metastasis, primary and secondary effectiveness rates, and major complication rates were recorded. Estimated glomerular filtration rates (GFRs) between pre-RFA and last follow-up were compared using paired t tests. The 2-year recurrence-free survival rate was calculated using Kaplan-Meier survival analysis. Of the 51 patients, 2 (3.9%) experienced local tumor progression, and 1 (2.0%) had lymph node metastasis after the first RFA session. Primary and secondary effectiveness rates were 96.1% (49/51) and 100% (1/1), respectively. Only 1 patient experienced a major complication (uretero-pelvic stricture) after the second RFA session for treating a local tumor progression, and the major complication rate was 1.9% (1/52). The median pre-RFA and last follow-up GFRs were 87.1 mL/ min/1.73 m{sup 2} (14.2–142.7 mL/min/1.73 m{sup 2}) and 72.0 mL/min/1.73 m{sup 2} (7.2–112.6 mL/min/1.73 m{sup 2}), respectively (p < 0.0001). The 2-year recurrence-free survival rate was 96.0%. CT-guided RFA is a safe and effective treatment in Korean patients with T1a RCC because of excellent mid-term outcomes.

  12. CT-guided radiofrequency ablation of T1a renal cell carcinma in Korea; Mid-erm outcomes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hae Jin; Park, Byung Kwan; Park, Jung Jae; Kim, Chan Kyo [Dept. of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2016-09-15

    To evaluate the mid-term outcomes of percutaneous radiofrequency ablation (RFA) treatment in patients with small (< 4 cm) renal cell carcinoma (RCC) in Korea. Between 2010 and 2015, 51 patients (40 men and 11 women; median age, 57 years) with biopsyproven 51 RCC were treated using CT-guided RFA. All patients were clinically staged T1aN0M0 prior to RFA. The median tumor size and follow-up period were 2.1 cm (range, 1.0–3.9 cm) and 26 months (4–60 months), respectively. Local tumor progression, distant metastasis, primary and secondary effectiveness rates, and major complication rates were recorded. Estimated glomerular filtration rates (GFRs) between pre-RFA and last follow-up were compared using paired t tests. The 2-year recurrence-free survival rate was calculated using Kaplan-Meier survival analysis. Of the 51 patients, 2 (3.9%) experienced local tumor progression, and 1 (2.0%) had lymph node metastasis after the first RFA session. Primary and secondary effectiveness rates were 96.1% (49/51) and 100% (1/1), respectively. Only 1 patient experienced a major complication (uretero-pelvic stricture) after the second RFA session for treating a local tumor progression, and the major complication rate was 1.9% (1/52). The median pre-RFA and last follow-up GFRs were 87.1 mL/ min/1.73 m{sup 2} (14.2–142.7 mL/min/1.73 m{sup 2}) and 72.0 mL/min/1.73 m{sup 2} (7.2–112.6 mL/min/1.73 m{sup 2}), respectively (p < 0.0001). The 2-year recurrence-free survival rate was 96.0%. CT-guided RFA is a safe and effective treatment in Korean patients with T1a RCC because of excellent mid-term outcomes.

  13. Targeting FLT3 in primary MLL-gene-rearranged infant acute lymphoblastic leukemia.

    Science.gov (United States)

    Stam, Ronald W; den Boer, Monique L; Schneider, Pauline; Nollau, Peter; Horstmann, Martin; Beverloo, H Berna; van der Voort, Ella; Valsecchi, Maria G; de Lorenzo, Paola; Sallan, Stephen E; Armstrong, Scott A; Pieters, Rob

    2005-10-01

    Acute lymphoblastic leukemia (ALL) in infants is characterized by rearrangements of the mixed lineage leukemia (MLL) gene, drug resistance, and a poor treatment outcome. Therefore, novel therapeutic strategies are needed to improve prognosis. Recently, we showed that FLT3 is highly expressed in MLL rearranged ALL (MLL). Here we demonstrate FLT3 expression in infants with MLL (n = 41) to be significantly higher compared to both infant (n = 8; P < .001) and noninfant patients with ALL (n = 23; P = .001) carrying germline MLL genes. Furthermore, leukemic cells from infants with MLL were significantly more sensitive to the Fms-like tyrosine kinase 3 (FLT3) inhibitor PKC412 (N-benzoyl staurosporine) than noninfant ALL cells, and at least as sensitive as internal tandem duplication-positive (ITD+) AML cells. Surprisingly, activation loop mutations only occurred in about 3% (1 of 36) of the cases and no FLT3/ITDs were observed. However, measuring FLT3 phosphorylation in infants with MLL expressing varying levels of wild-type FLT3 revealed that high-level FLT3 expression is associated with ligand-independent FLT3 activation. This suggests that infant MLL cells displaying activated FLT3 as a result of overexpression can be targeted by FLT3 inhibitors such as PKC412. However, at concentrations of PKC412 minimally required to fully inhibit FLT3 phosphorylation, the cytotoxic effects were only fractional. Thus, PKC412-induced apoptosis in infant MLL cells is unlikely to be a consequence of FLT3 inhibition alone but may involve inhibition of multiple other kinases by this drug.

  14. Rhabdoid Variant of Myoepithelial Carcinoma, with EWSR1 Rearrangement: Expanding the Spectrum of EWSR1-Rearranged Myoepithelial Tumors.

    Science.gov (United States)

    Thway, Khin; Bown, Nick; Miah, Aisha; Turner, Rob; Fisher, Cyril

    2015-06-01

    Myoepithelial and mixed tumors represent a heterogeneous group of neoplasms for which classification is incomplete and continues to evolve. Those arising in the soft tissues appear to represent subgroups that are genetically distinct from those that occur within salivary glands. We describe a case of soft tissue myoepithelial carcinoma with rhabdoid morphology, which presented as an enlarging neck mass in a 40 year old male, and in which EWSR1 rearrangement was demonstrated by fluorescence in situ hybridization. This neoplasm showed diffuse INI1 loss, making distinction from other INI1-negative rhabdoid tumors difficult. This expands the range of reported histologic features of EWSR1-rearranged myoepithelial neoplasms, and highlights the significant morphologic and immunohistochemical overlap between this and other INI1-negative malignant rhabdoid neoplasms.

  15. Blood flow changes coincide with cellular rearrangements during blood vessel pruning in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Eva Kochhan

    Full Text Available After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y-shaped structure. Subsequently, endothelial cells in parts of the previously perfused branch rearrange from a multicellular into a unicellular tube, followed by blood vessel detachment. This process is accompanied by endothelial cell death. Finally, we show that differences in blood flow between neighboring vessels are important for the completion of the pruning process. Our data suggest that flow induced changes in tubular architecture ensure proper blood vessel pruning.

  16. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect.

    Science.gov (United States)

    Luo, Lihua; Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan; Wang, Xiong; Huselstein, Celine; Chen, Yun

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  17. Development of a multi-step leukemogenesis model of MLL-rearranged leukemia using humanized mice.

    Directory of Open Access Journals (Sweden)

    Kunihiko Moriya

    Full Text Available Mixed-lineage-leukemia (MLL fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/- (NOG mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification. We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

  18. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene].

    Science.gov (United States)

    Oh, Hye Ryong; Lee, Mi Ja; Park, Geon; Moon, Dae Soo; Park, Young Jin; Jang, Sook Jin

    2009-06-01

    A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

  19. Imperfect conformation of experimental and epidemiological data for frequency of RET/РТС gene rearrangements in papillary thyroid carcinoma for the Chernobyl accident

    Directory of Open Access Journals (Sweden)

    Ushenkova L.N.

    2013-12-01

    Full Text Available In an overview and analytical study of the epidemiological data on the frequency of RET/РТС gene rearrangements in sporadic and radiogenic (patients after radiotherapy, residents of contaminated after the Chernobyl disaster areas, victims after the atomic bombings, etc. carcinomas of the thyroid gland were examined. In general, the observed epidemiological laws were confirmed in radiobiology experiments by irradiation of different cultures of thyroid cells and ex vivo with the exception of Chernobyl cohorts. Induction of RET/РТС gene rearrangements by 131l exposure in children carcinomas of Chernobyl residents in mice did not observe too. It is concluded that the situation with the frequency of RET/РТС rearrangements in thyroid carcinoma in Chernobyl cohorts once again confirms the multifactorial nature of the induction and development of these tumors with a contribution of radiation and non-radiation factors (iodine deficiency and different stresses.

  20. Tumor Control Outcomes After Hypofractionated and Single-Dose Stereotactic Image-Guided Intensity-Modulated Radiotherapy for Extracranial Metastases From Renal Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zelefsky, Michael J., E-mail: zelefskm@mskcc.org [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Greco, Carlo [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Motzer, Robert [Solid Tumor Service, Medical Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Magsanoc, Juan Martin; Pei Xin [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Lovelock, Michael; Mechalakos, Jim [Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Zatcky, Joan; Fuks, Zvi; Yamada, Yoshiya [Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

    2012-04-01

    Purpose: To report tumor local progression-free outcomes after treatment with single-dose, image-guided, intensity-modulated radiotherapy and hypofractionated regimens for extracranial metastases from renal cell primary tumors. Patients and Methods: Between 2004 and 2010, 105 lesions from renal cell carcinoma were treated with either single-dose, image-guided, intensity-modulated radiotherapy to a prescription dose of 18-24 Gy (median, 24) or hypofractionation (three or five fractions) with a prescription dose of 20-30 Gy. The median follow-up was 12 months (range, 1-48). Results: The overall 3-year actuarial local progression-free survival for all lesions was 44%. The 3-year local progression-free survival for those who received a high single-dose (24 Gy; n = 45), a low single-dose (<24 Gy; n = 14), or hypofractionation regimens (n = 46) was 88%, 21%, and 17%, respectively (high single dose vs. low single dose, p = .001; high single dose vs. hypofractionation, p < .001). Multivariate analysis revealed the following variables were significant predictors of improved local progression-free survival: 24 Gy dose compared with a lower dose (p = .009) and a single dose vs. hypofractionation (p = .008). Conclusion: High single-dose, image-guided, intensity-modulated radiotherapy is a noninvasive procedure resulting in high probability of local tumor control for metastatic renal cell cancer generally considered radioresistant according to the classic radiobiologic ranking.

  1. Characterization of cryptic rearrangements, deletion, complex variants of PML, RARA in acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    Pratibha Kadam Amare

    2011-01-01

    Full Text Available Acute promyelocytic leukemia (APL is characterized by a reciprocal translocation t(15;17(q22;q21 leading to the disruption of Promyelocytic leukemia (PML and Retionic Acid Receptor Alpha (RARA followed by reciprocal PML-RARA fusion in 90% of the cases. Fluorescence in situ hybridization (FISH has overcome the hurdles of unavailability of abnormal and/or lack of metaphase cells, and detection of cryptic, submicroscopic rearrangements. In the present study, besides diagnostic approach we sought to analyze these cases for identification and characterization of cryptic rearrangements, deletion variants and unknown RARA translocation variants by application of D-FISH and RARA break-apart probe strategy on interphase and metaphase cells in a large series of 200 cases of APL. Forty cases (20% had atypical PML-RARA and/or RARA variants. D-FISH with PML/RARA probe helped identification of RARA insertion to PML. By application of D-FISH on metaphase cells, we documented that translocation of 15 to 17 leads to 17q deletion which results in loss of reciprocal fusion and/or residual RARA on der(17. Among the complex variants of t(15;17, PML-RARA fusion followed by residual RARA insertion closed to PML-RARA on der(15 was unique and unusual. FISH with break-apart RARA probe on metaphase cells was found to be a very efficient strategy to detect unknown RARA variant translocations like t(11;17(q23;q21, t(11;17(q13;q21 and t(2;17(p21;q21. These findings proved that D-FISH and break-apart probe strategy has potential to detect primary as well as secondary additional aberrations of PML, RARA and other additional loci. The long-term clinical follow-up is essential to evaluate the clinical importance of these findings.

  2. Gyrokinetic and kinetic particle-in-cell simulations of guide-field reconnection. Part I: macroscopic effects of the electron flows

    CERN Document Server

    Muñoz, P A; Kilian, P; Büchner, J; Jenko, F

    2015-01-01

    In this work, we extend a comparison between gyrokinetic (GK) and fully kinetic Particle-in-Cell (PIC) simulations of magnetic reconnection in the limit of strong guide field started by TenBarge et al. [Phys. Plasmas 21, 020708 (2014)]. By using a different set of kinetic PIC and GK simulation codes (ACRONYM and GENE, respectively), we analyze the limits of applicability of the GK approach when comparing to the force free kinetic simulations in the low guide field (bg) regime. Here we report the first part of a much more extended comparison, focusing on the macroscopic effects of the electron flows. For a low beta plasma (beta_i = 0.01), it is shown that magnetic reconnection only displays similar features between both plasma models for higher kinetic PIC guide fields (bg>30) in the secondary magnetic islands than in the region close to the X points or separatrices (bg>5). Kinetic PIC low guide field runs (53) to be negligible due to the reduced reconnection rate and fluctuation level.

  3. Use of laboratory tests to guide initiation of autologous hematopoietic progenitor cell collection by apheresis: results from the multicenter hematopoietic progenitor cell collection by Apheresis Laboratory Trigger Survey.

    Science.gov (United States)

    Makar, Robert S; Padmanabhan, Anand; Kim, Haewon C; Anderson, Christina; Sugrue, Michele W; Linenberger, Michael

    2014-10-01

    Limited literature describes the value of laboratory "triggers" to guide collection of peripheral blood (PB) hematopoietic progenitor cells (HPCs) by apheresis [HPC(A)]. We used a web-based survey to determine which parameters are used to initiate autologous HPC(A) collection in adult and pediatric patients and to identify common practice patterns. Members of the AABB Cellular Therapy Product Collection and Clinical Practices Subsection and the American Society for Apheresis HPC Donor Subcommittee drafted and developed relevant survey questions. A web link to the survey was distributed by electronic newsletter or email. Responses from 67 programs that perform autologous HPC(A) collections, including academic medical centers (n = 46), blood centers (n = 10), community hospitals (n = 5), and a variety of other medical institutions (n = 6), were analyzed. Ninety-three percent (62/67) of programs used a laboratory parameter to initiate HPC(A) collection. In both adult (40/54, 74%) and pediatric (29/38, 76%) patients, the PB CD34+ cell count was the most common parameter used to initiate HPC(A) collection. The median PB CD34+ trigger value was 10/μL for both patient populations. Among centers routinely using the PB CD34+ cell count to initiate apheresis, 51% (22/43) first sent the test before the patient presented for collection. Although more than 90% of centers used a laboratory test to trigger apheresis in cytokine-mobilized (44/48) or chemomobilized patients (50/53), only 57% (30/53) used a laboratory trigger if the patient was mobilized with granulocyte colony-stimulating factor plus plerixafor. Forty-two percent (21/50) of programs that routinely measured the PB CD34+ count before collection and discontinued further HPC(A) collection based on product CD34+ cell yield also stopped if the PB CD34+ value before apheresis was considered too low to proceed. Most programs use the PB CD34+ cell count to trigger autologous HPC(A) collection. Some centers also use this

  4. Metastatic renal cell carcinoma from a native kidney of a renal transplant patient diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA biopsy

    Directory of Open Access Journals (Sweden)

    Yaseen Alastal

    2015-04-01

    Full Text Available Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA biopsy sampling of enlarged lymph nodes is increasingly used to diagnose metastatic tumors, especially of the gastrointestinal tract and the lungs. Herein, we describe the diagnosis of metastatic renal cell carcinoma from a native kidney of a 54 year-old male patient, who had a 5-years history of renal transplant, by EUS-FNA of mediastinal and celiac lymph nodes. Histological and immunohistochemical findings confirmed the origin of metastatic tumor. EUS-FNA with proper cytological evaluation can be useful in the diagnosis of metastatic renal cell carcinoma in renal transplant patients. 

  5. Cytogenetic, FISH and molecular characterization of 3q27/BCL-6 rearrangements in NHL

    Energy Technology Data Exchange (ETDEWEB)

    Wiodarska, I.; Styl, M.; Mecucci, C. [Univ. of Leuven (Belgium)] [and others

    1994-09-01

    Reciprocal translocations involving the chromosomal region 3q27 and one of the immunoglobulin loci at 14q32, 2p12 or 22q11 have been identified as the third most common type of chromosomal abnormality in Non Hodgkin`s lymphomas (NHLs), in addition to t(14;18) and t(8;14). These abnormalities appeared to be strongly associated with a diffuse, large cell subtype of B-cell NHL. Recently, a t(3;14) and t(3;22) have been cloned and a new transcriptional unit at 3q27, designated BCL-5, BCL-6 or LAZ3, has been identified. The gene appears to encode a new zinc finger protein with the putative function of a transcription factor. Rearrangements of the BCL-6 gene have been detected not only in cases with a typical t(3;14), t(2;3) and t(3;22), but also in a few NHL cases carrying 3q27 translocations not involving Ig genes. We report on nine B-NHL cases with a 3q27/BCL-6 rearrangement demonstrated by cytogenetic, FISH, and Southern analysis. Cytogenetic analysis complemented by FISH studies showed the presence of a classical t(3;14) or a t(3;22) in three cases and a variety of chromosomal aberrations involving the 3q27 locus in the remaining cases. Some of these translocations were not previously identified by conventional banding analysis. In three patients chromosome painting demonstrated involvement of both chromosome at the 3q24 band. We conclude: 3q27/BCL-6 rearrangements seem not to be restricted to diffuse large cell lymphoma. We here documented 3q27/BCL-6 abnormalities in Richter syndrome and follicular lymphomas. The variety of 3q27 aberrations at cytogenetic level suggests that, in addition to immunoglobulin genes, a number of other genes spreading over the human genome may deregulate BCL-6 in lymphomas. Chromosome painting is a powerful tool to demonstrate 3q27 abnormalities, not identified by conventional banding analysis.

  6. Rearrangements of microtubule cytoskeleton in stomatal closure of Arabidopsis induced by nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG YongMei; WU ZhongYi; WANG XueChen; YU Rong

    2008-01-01

    NO (nitric oxide), known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows: (ⅰ) In vivo stomatal aperture assays revealed that both vinblastine (microtubule-disrupting drug) and SNP (exogenous NO donor) prevented stomatal opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas taxol (a microtubule-stabilizing agent) was able to reduce this effect; (ⅱ) microtubules in the opening Arabi-dopsis guard cells expressing GFP:α-tubulin-6 (AtGFP:α-tubulin-6) were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; (ⅲ) under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and be-came oblique or exhibited a random pattern; (ⅳ) furthermore, the involvement of cytosolic Ca2+ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM (a chelator of Ca2+) greatly sup-pressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of [Ca2+]cyt (free Ca2+ concentration in the cy-toplasm), finally leading to stomatal closure.

  7. Refining borders of genome-rearrangements including repetitions

    Directory of Open Access Journals (Sweden)

    JA Arjona-Medina

    2016-10-01

    Full Text Available Abstract Background DNA rearrangement events have been widely studied in comparative genomic for many years. The importance of these events resides not only in the study about relatedness among different species, but also to determine the mechanisms behind evolution. Although there are many methods to identify genome-rearrangements (GR, the refinement of their borders has become a huge challenge. Until now no accepted method exists to achieve accurate fine-tuning: i.e. the notion of breakpoint (BP is still an open issue, and despite repeated regions are vital to understand evolution they are not taken into account in most of the GR detection and refinement methods. Methods and results We propose a method to refine the borders of GR including repeated regions. Instead of removing these repetitions to facilitate computation, we take advantage of them using a consensus alignment sequence of the repeated region in between two blocks. Using the concept of identity vectors for Synteny Blocks (SB and repetitions, a Finite State Machine is designed to detect transition points in the difference between such vectors. The method does not force the BP to be a region or a point but depends on the alignment transitions within the SBs and repetitions. Conclusion The accurate definition of the borders of SB and repeated genomic regions and consequently the detection of BP might help to understand the evolutionary model of species. In this manuscript we present a new proposal for such a refinement. Features of the SBs borders and BPs are different and fit with what is expected. SBs with more diversity in annotations and BPs short and richer in DNA replication and stress response, which are strongly linked with rearrangements.

  8. MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.

    Directory of Open Access Journals (Sweden)

    Georg Dössinger

    Full Text Available Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs, we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN. Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25% and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+ T cells without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.

  9. Thermal rearrangement of novolak resins used in microlithography

    Science.gov (United States)

    Hardy, Ricky; Zampini, Anthony; Monaghan, Michael J.; O'Leary, Michael J.; Cardin, William J.; Eugster, Timothy J.

    1995-06-01

    Changes in phenolic-formaldehyde resin properties are described in terms of thermal exposure. At high temperature, resin molecular weight, dissolution properties and chemical composition change depending on the presence or absence of monomers. Without monomer in the resin melt at 220 degree(s)C, resin molecular weight increases with a corresponding decrease in dissolution rate. In the presence of monomer, molecular weight generally decreases. Dissolution rate may fluctuate depending on the monomer mixture. Three,five- Xylenol and 2,3,5-trimethylphenol co-monomers induced the most extreme changes in resin properties with thermal treatment. Resin degradation-recombination processes suggest a classical Friedel-Craft rearrangement mechanism.

  10. [Clonality lymphoid study through rearrangement analysis of antigen receptor].

    Science.gov (United States)

    Villamizar-Rivera, Nicolás; Olaya, Natalia

    2015-01-01

    As a rule, malignant lymphoid proliferations are clonal. While most of the time the biological potential can be established through routine pathologic examination and auxiliary techniques, some cases are difficult to classify. Moreover, there are situations in which there are dominant clones whose analysis are important, such as occur in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality in lymphoid lesions, i.e. the analysis of rearrangements of antigen receptor genes by multiplex polymerase chain reaction (PCR) based tests.

  11. Confining Bond Rearrangement in the Random Center Vortex Model

    CERN Document Server

    Altarawneh, Derar; Engelhardt, Michael

    2015-01-01

    We present static meson-meson and baryon--anti-baryon potentials in Z(2) and Z(3) random center vortex models for the infrared sector of Yang-Mills theory, i.e., hypercubic lattice models of random vortex world-surfaces. In particular, we calculate Polyakov loop correlators of two static mesons resp. (anti-)baryons in a center vortex background and observe that their expectation values follow the minimal area law and show bond rearrangement behavior. The static meson-meson and baryon--anti-baryon potentials are compared with theoretical predictions and lattice QCD simulations.

  12. Precise detection of rearrangement breakpoints in mammalian chromosomes

    Directory of Open Access Journals (Sweden)

    Gautier Christian

    2008-06-01

    Full Text Available Abstract Background Genomes undergo large structural changes that alter their organisation. The chromosomal regions affected by these rearrangements are called breakpoints, while those which have not been rearranged are called synteny blocks. We developed a method to precisely delimit rearrangement breakpoints on a genome by comparison with the genome of a related species. Contrary to current methods which search for synteny blocks and simply return what remains in the genome as breakpoints, we propose to go further and to investigate the breakpoints themselves in order to refine them. Results Given some reliable and non overlapping synteny blocks, the core of the method consists in refining the regions that are not contained in them. By aligning each breakpoint sequence against its specific orthologous sequences in the other species, we can look for weak similarities inside the breakpoint, thus extending the synteny blocks and narrowing the breakpoints. The identification of the narrowed breakpoints relies on a segmentation algorithm and is statistically assessed. Since this method requires as input synteny blocks with some properties which, though they appear natural, are not verified by current methods for detecting such blocks, we further give a formal definition and provide an algorithm to compute them. The whole method is applied to delimit breakpoints on the human genome when compared to the mouse and dog genomes. Among the 355 human-mouse and 240 human-dog breakpoints, 168 and 146 respectively span less than 50 Kb. We compared the resulting breakpoints with some publicly available ones and show that we achieve a better resolution. Furthermore, we suggest that breakpoints are rarely reduced to a point, and instead consist in often large regions that can be distinguished from the sequences around in terms of segmental duplications, similarity with related species, and transposable elements. Conclusion Our method leads to smaller

  13. Guided labworks

    DEFF Research Database (Denmark)

    Jacobsen, Lærke Bang

    For the last 40 years physics education research has shown poor learning outcomes of guided labs. Still this is found to be a very used teaching method in the upper secodary schools. This study explains the teacher's choice of guided labs throught the concept of redesign as obstacle dislodgement...

  14. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Lihua [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Center of Molecular Medicine, School of Medicine, Hubei University of Arts and Sciences, Xiangyang 441053 (China); Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Wang, Xiong; Huselstein, Celine [Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS – Université de Lorraine, Biopôle, 54500 Vandoeuvre-lès-Nancy (France); Chen, Yun, E-mail: yunchen@whu.edu.cn [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  15. Differential regulation of the c-Myc/Lin28 axis discriminates subclasses of rearranged MLL leukemia

    Science.gov (United States)

    Chen, Lili; Sun, Yuqing; Wang, Jingya; Jiang, Hui; Muntean, Andrew G.

    2016-01-01

    MLL rearrangements occur in myeloid and lymphoid leukemias and are generally associated with a poor prognosis, however this varies depending on the fusion partner. We modeled acute myeloid leukemia (AML) in mice using various MLL fusion proteins (MLL-FPs) and observed significantly different survival outcomes. To better understand the differences between these leukemias, we examined the genome wide expression profiles of leukemic cells transformed with different MLL-FPs. RNA-sequencing and pathway analysis identified the c-Myc transcriptional program as one of the top distinguishing features. c-Myc protein levels were highly correlative with AML disease latency in mice. Functionally, overexpression of c-Myc resulted in a more aggressive proliferation rate in MLL-FP cell lines. While all MLL-FP transformed cells displayed sensitivity to BET inhibitors, high c-Myc expressing cells showed greater resistance to Brd4 inhibition. The Myc target Lin28B was also differentially expressed in MLL-FP cell lines in agreement with c-Myc expression. Examination of Lin28B miRNAs targets revealed that let-7g was significantly increased in leukemic cells associated with the longest disease latency and forced let-7g expression induced differentiation of leukemic blasts. Thus, differential regulation of the c-Myc/Lin28/let-7g program by different MLL-FPs is functionally related to disease latency and BET inhibitor resistance in MLL leukemias. PMID:27007052

  16. Constitutional Chromothripsis Rearrangements Involve Clustered Double-Stranded DNA Breaks and Nonhomologous Repair Mechanisms

    Directory of Open Access Journals (Sweden)

    Wigard P. Kloosterman

    2012-06-01

    Full Text Available Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements.

  17. Bio-guided isolation of the cytotoxic terpenoids from the roots of Euphorbia kansui against human normal cell lines L-O2 and GES-1.

    Science.gov (United States)

    Zhang, Li; Gao, Lan; Li, Zhengjun; Yan, Xiaojing; Yang, Yanjing; Tang, Yuping; Cao, Yudan; Ding, Anwei

    2012-01-01

    The dried roots of Euphorbia kansui (kansui) have been used for centuries in China as a herbal medicine for edema, ascites, and asthma. The 95% ethanol extract showed a significant inhibition of cell proliferation against human normal cell lines L-O2 and GES-1. Bioassay-guided separation of the 95% ethanol extract from the roots of E. kansui led to the isolation of 12 diverse terpenoids whose structures were identified by (1)H, (13)C NMR spectroscopy and ESI-MS as kansuinine A (1), kansuinine B (2), kansuinine C (3), kansuiphorin C (4), 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (5), 3-O-(2'E,4'Edecadienoyl)-20-O-acetylingenol (6), 3-O-(2'E,4'Z-decadienoyl)-20-deoxyingenol (7), 3-O-benzoyl-20-deoxyingenol (8), 5-O-benzoyl-20-deoxyingenol (9), kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5-11) exhibited a relatively lower IC(50) value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb.

  18. Epigenetic and 3-dimensional regulation of V(D)J rearrangement of immunoglobulin genes.

    Science.gov (United States)

    Degner-Leisso, Stephanie C; Feeney, Ann J

    2010-12-01

    V(D)J recombination is a crucial component of the adaptive immune response, allowing for the production of a diverse antigen receptor repertoire (Ig and TCR). This review will focus on how epigenetic regulation and 3-dimensional (3D) interactions may control V(D)J recombination at Ig loci. The interplay between transcription factors and post-translational modifications at the Igh, Igκ, and Igλ loci will be highlighted. Furthermore, we propose that the spatial organization and epigenetic boundaries of each Ig loci before and during V(D)J recombination may be influenced in part by the CTCF/cohesin complex. Taken together, the many epigenetic and 3D layers of control ensure that Ig loci are only rearranged at appropriate stages of B cell development.

  19. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  20. Transposon domestication versus mutualism in ciliate genome rearrangements.

    Directory of Open Access Journals (Sweden)

    Alexander Vogt

    Full Text Available Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.

  1. Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase.

    Science.gov (United States)

    Bordes, Florence; Barbe, Sophie; Escalier, Pierre; Mourey, Lionel; André, Isabelle; Marty, Alain; Tranier, Samuel

    2010-10-06

    We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.

  2. Rearrangement mechanism of the sodium adducts of Fmoc protected amino acids

    Institute of Scientific and Technical Information of China (English)

    DU Jintang; LI Yanmei; ZHU Zhentai; CHEN Yi; ZHAO Yufen

    2003-01-01

    The cationized 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acidswere analyzed by the electrospray ionization tandem mass spectrometry (ESI-MS/MS). A rearrangement reaction leading to the C-terminal hydroxyl group transfer was observed. The sodium adducts of Fmoc-OH was formed. A possible rearrangement mechanism was proposed. The rearrangement reaction depended on the Fmoc group, metal ions and metal ion radius. It was shown that the Fmoc group has a strong affinity to the hydroxyl group in the gas phase.

  3. HBR guides

    CERN Document Server

    Duarte, Nancy; Dillon, Karen

    2015-01-01

    Master your most pressing professional challenges with this seven-volume set that collects the smartest best practices from leading experts all in one place. "HBR Guide to Better Business Writing" and "HBR Guide to Persuasive Presentations" help you perfect your communication skills; "HBR Guide to Managing Up and Across" and "HBR Guide to Office Politics" show you how to build the best professional relationships; "HBR Guide to Finance Basics for Managers" is the one book you'll ever need to teach you about the numbers; "HBR Guide to Project Management" addresses tough questions such as how to manage stakeholder expectations and how to manage uncertainty in a complex project; and "HBR Guide to Getting the Right Work Done" goes beyond basic productivity tips to teach you how to prioritize and focus on your work. This specially priced set of the most popular books in the series makes a perfect gift for aspiring leaders looking for trusted advice. Arm yourself with the advice you need to succeed on the job, from ...

  4. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    Science.gov (United States)

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  5. The spectrum of EWSR1-rearranged neoplasms at a tertiary sarcoma centre; assessing 772 tumour specimens and the value of current ancillary molecular diagnostic modalities

    Science.gov (United States)

    Noujaim, Jonathan; Jones, Robin L; Swansbury, John; Gonzalez, David; Benson, Charlotte; Judson, Ian; Fisher, Cyril; Thway, Khin

    2017-01-01

    Background: EWSR1 rearrangements were first identified in Ewing sarcoma, but the spectrum of EWSR1-rearranged neoplasms now includes many soft tissue tumour subtypes including desmoplastic small round cell tumour (DSRCT), myxoid liposarcoma (MLPS), extraskeletal myxoid chondrosarcoma (EMC), angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma (CCS) and myoepithelial neoplasms. We analysed the spectrum of EWSR1-rearranged soft tissue neoplasms at our tertiary sarcoma centre, by assessing ancillary molecular diagnostic modalities identifying EWSR1-rearranged tumours and reviewing the results in light of our current knowledge of these and other Ewing sarcoma-like neoplasms. Methods: We retrospectively analysed all specimens tested for EWSR1 rearrangements by fluorescence in situ hybridisation (FISH) and/or reverse transcription–PCR (RT–PCR) over a 7-year period. Results: There was a total of 772 specimens. FISH was performed more often than RT–PCR (n=753, 97.5% vs n=445, 57.6%). In total, 210 (27.9%) specimens were FISH-positive for EWSR1 rearrangement compared to 111 (14.4%) that showed EWSR1 fusion transcripts with RT–PCR. Failure rates for FISH and RT–PCR were 2.5% and 18.0%. Of 109 round cell tumours with pathology consistent with Ewing sarcoma, 15 (13.8 %) cases were FISH-positive without an identifiable EWSR1 fusion transcript, 4 (3.7%) were FISH-negative but RT–PCR positive and 4 (3.7%) were negative for both. FISH positivity for DSRCT, MLPS, EMC, AFH and CCS was 86.3%, 4.3%, 58.5%, 60.0% and 87.9%, respectively. A positive FISH result led to diagnostic change in 40 (19.0%) EWSR1-rearranged cases. 13 FISH-positive cases remained unclassifiable. Conclusions: FISH is more sensitive for identifying EWSR1 rearrangements than RT–PCR. However, there can be significant morphologic and immunohistochemical overlap between groups of EWSR1-rearranged neoplasms, with important prognostic and therapeutic implications. FISH and RT–PCR should be used as

  6. Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury.

    Science.gov (United States)

    Minns, Martin S; Teicher, Gregory; Rich, Celeste B; Trinkaus-Randall, Vickery

    2016-02-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein-labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing.

  7. Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

    Science.gov (United States)

    Minns, Martin S.; Teicher, Gregory; Rich, Celeste B.; Trinkaus-Randall, Vickery

    2017-01-01

    The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein–labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. PMID:26683661

  8. The prognostic index: a useful pathologic guide for prediction of nodal metastases and survival in penile squamous cell carcinoma.

    Science.gov (United States)

    Chaux, Alcides; Caballero, Carmelo; Soares, Fernando; Guimarães, Gustavo C; Cunha, Isabel W; Reuter, Víctor; Barreto, José; Rodríguez, Ingrid; Cubilla, Antonio L

    2009-07-01

    A concern of surgical oncologists has been to find a method to select patients for groin dissection in penile carcinomas considering the high morbidity of this procedure. A promising methodology, in the identification of early metastatic foci by the sentinel lymph node technique (initiated in Paraguay in the 1970s), was found, using a static anatomic approach, to be associated with a recurrence rate of 30%. Later, a dynamic method using radioactive tracers and peritumoral dye injection was introduced with an improvement in patients' outcome. Recurrences, however, remained high in most studies at a rate of about 15% to 20% except in few highly specialized centers with failure rates of 5%. The technical sophistication, lack of multicenter reproduction, and cost of dynamic sentinel node biopsies preclude their routine implementation in developing countries and other approaches are necessary. Because histologic grade, depth of tumor infiltration, and perineural invasion (PNI) are considered among the most important pathologic prognostic parameters in penile cancer, we devised a Prognostic Index combining these 3 factors. In this study, we are evaluating the incidence of nodal metastasis according to the Prognostic Index score. Pathologic materials from 193 patients with penectomy/circumcision and bilateral groin dissections for invasive squamous cell carcinoma were analyzed. The Prognostic Index (ranging from 2 to 7) consisted in the addition of numerical values given to histologic grade (1 to 3), deepest anatomic level involved by cancer (1 to 3), and presence of PNI (0 or 1). Histologic grades were defined as follows: grade 1, carcinomas with minimal to no atypias; grade 3, tumors showing any proportion of anaplastic cells; and grade 2, the remainder tumors. The anatomic levels and their numerical values were: in glans, lamina propria, 1; corpus spongiosum, 2; and corpus cavernosum, 3. In foreskin they were: lamina propria, 1; dartos, 2; and skin, 3. PNI was

  9. Antiproliferative activity and induction of apoptosis in PC-3 cells by the chalcone cardamonin from Campomanesia adamantium (Myrtaceae) in a bioactivity-guided study.

    Science.gov (United States)

    Pascoal, Aislan Cristina Rheder Fagundes; Ehrenfried, Carlos Augusto; Lopez, Begoña Gimenez-Cassina; de Araujo, Thiago Matos; Pascoal, Vinicius D'ávila Bitencourt; Gilioli, Rovilson; Anhê, Gabriel Forato; Ruiz, Ana Lúcia Tasca Goes; Carvalho, João Ernesto de; Stefanello, Maria Elida Alves; Salvador, Marcos José

    2014-02-07

    The Myrtaceae family is a common source of medicines used in the treatment of numerous diseases in South America. In Brazil, fruits of the Campomanesia species are widely used to make liqueurs, juices and sweets, whereas leaves are traditionally employed as a medicine for dysentery, stomach problems, diarrhea, cystitis and urethritis. Ethanol extracts of Campomanesia adamantium (Myrtaceae) leaves and fruits were evaluated against prostate cancer cells (PC-3). The compound (2E)-1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylprop-2-en-1-one, cardamonin) was isolated from ethanol extracts of C. adamantium leaves in a bioactivity-guided study and quantified by UPLC-MS/MS. In vitro studies showed that the isolated chalcone cardamonin inhibited prostate cancer cell proliferation and decreased the expression of NFkB1. Moreover, analysis by flow cytometry showed that this compound induced DNA fragmentation, suggesting an effect on apoptosis induction in the PC-3 cell line.

  10. Assessment of RET/PTC1 and RET/PTC3 rearrangements in fine-needle aspiration biopsy specimens collected from patients with Hashimoto's thyroiditis

    Directory of Open Access Journals (Sweden)

    Cyniak-Magierska Anna

    2011-01-01

    Full Text Available Abstract Background RET/PTC rearrangements are the most frequent molecular changes in papillary thyroid carcinoma (PTC. So far, 15 main RET/PTC rearrangements have been described, among which RET/PTC1 and RET/PTC3 are the most common in PTC - especially in radiation-induced tumours. RET/PTC1 and RET/PTC3 are the result of intrachromosomal paracentric inversions in chromosome 10, where RET and the activating genes (H4 and ELE1, respectively are located. Recently, RET/PTC rearrangements have been shown not only in PTC but also in benign thyroid lesions, including Hashimoto's thyroiditis (HT. The aim of study was an assessment of RET/PTC1 and RET/PTC3 rearrangements in patients with Hashimoto's thyroiditis. Materials and methods Thyroid aspirates, eligible for the study, were obtained from 26 patients with Hashimoto's thyroiditis by fine-needle aspiration biopsy (FNAB. Each aspirate was smeared for conventional cytology, while its remaining part was immediately washed out of the needle. The cells, obtained from the needle, were used in further investigation. Total RNA from FNAB was extracted by use of an RNeasy Micro Kit, based on modified Chomczynski and Sacchi's method and reverse transcription (RT-PCR was done. Quantitative evaluation of RET/PTC1 and RET/PTC3 rearrangements by real-time PCR was performed by an ABI PRISM® 7500 Sequence Detection System. In the study, PTC tissues with known RET/PTC1 and RET/PTC3 rearrangements served as a reference standard (calibrator, while β-actin gene was used as endogenous control. Results Amplification reactions were done in triplicate for each examined sample. No RET/PTC1 and RET/PTC3 rearrangements were found in the examined samples. Conclusions Our results indicate that RET/PTC1 and RET/PTC3 rearrangements in Hashimoto's thyroiditis, if any, are rather rare events and further investigations should be conducted in order to determine molecular changes, connecting Hashimoto's thyroiditis with PTC.

  11. Colostomy Guide

    Science.gov (United States)

    ... Side Effects Managing Cancer-related Side Effects Ostomies Colostomy Guide Colostomy surgery is done for many different diseases and problems. Some colostomies are done because of cancer; others are not. ...

  12. Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris.

    Science.gov (United States)

    Diluvio, Laura; Vollmer, Sigrid; Besgen, Petra; Ellwart, Joachim W; Chimenti, Sergio; Prinz, Joerg C

    2006-06-01

    Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.

  13. CCR7 guides migration of mesenchymal stem cell to secondary lymphoid organs: a novel approach to separate GvHD from GvL effect.

    Science.gov (United States)

    Li, Hong; Jiang, YanMing; Jiang, XiaoXia; Guo, XiMin; Ning, HongMei; Li, YuHang; Liao, Li; Yao, HuiYu; Wang, XiaoYan; Liu, YuanLin; Zhang, Yi; Chen, Hu; Mao, Ning

    2014-07-01

    Inefficient homing of systemically infused mesenchymal stem cells (MSCs) limits the efficacy of existing MSC-based clinical graft-versus-host disease (GvHD) therapies. Secondary lymphoid organs (SLOs) are the major niches for generating immune responses or tolerance. MSCs home to a wide range of organs, but rarely to SLOs after intravenous infusion. Thus, we hypothesized that targeted migration of MSCs into SLOs may significantly improve their immunomodulatory effect. Here, chemokine receptor 7 (CCR7) gene, encoding a receptor that specifically guides migration of immune cells into SLOs, was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (MSCs/CCR7). We found that infusion of MSCs/CCR7 potently prolonged the survival of GvHD mouse model. The infused MSCs/CCR7 migrate to SLOs, relocate in proximity with T lymphocytes, therefore, potently inhibited their proliferation, activation, and cytotoxicity. Natural killer (NK) cells contribute to the early control of leukemia relapse. Although MSCs/CCR7 inhibited NK cell activity in vitro coculture, they did not impact on the proportion and cytotoxic capacities of NK cells in the peripheral blood of GvHD mice. In an EL4 leukemia cell loaded GvHD model, MSCs/CCR7 infusion preserved the graft-versus-leukemia (GvL) effect. In conclusion, this study demonstrates that CCR7 guides migration of MSCs to SLOs and thus highly intensify their in vivo immunomodulatory effect while preserving the GvL activity. This exciting therapeutic strategy may improve the clinical efficacy of MSC based therapy for immune diseases.

  14. A novel technique for guided bone regeneration using platelet-rich plasma and osteogenic progenitor cells: Literature-based rationale and case report.

    Science.gov (United States)

    Kwon, TaeHyun; Grieco, Peter C; Levin, Liran; Intini, Giuseppe

    2016-03-01

    Achieving predictable guided bone regeneration in critical size defects for future endosseous dental implant therapy poses a great challenge to clinicians. A novel technique utilizing autogenous osteogenic progenitor cells, calcium sulfate activated platelet-rich plasma in addition to particulate allograft was successfully used to augment a severely deficient maxillary anterior edentulous ridge. After 6 months of healing, satisfactory radiographic and clinical bone gain was noted with significant increase in alveolar ridge width. Endosseous implants were placed and restored successfully. The techniques with underlying clinical and biologic rationales are presented and discussed in this report.

  15. ERG rearrangement is associated with prostate cancer-related death in Chinese prostate cancer patients.

    Directory of Open Access Journals (Sweden)

    Mei Qi

    Full Text Available Recently, ETS-related gene (ERG gene rearrangements, phosphatase tensin homologue (PTEN deletions and EGFR family aberrations were characterized as potential biomarkers for prostate cancer (PCa patient management. Although ERG gene rearrangement has been identified in approximately 50% of localized prostate cancers in western countries, the prognostic significance of this critical molecular event remains unknown in Chinese patients. Using fluorescence in situ hybridization (FISH and immunohistochemistry, we evaluated ERG, PTEN and EGFR family aberrations in a cohort of 224 Chinese prostate cancer patients diagnosed in transurethral resection of the prostate (TUR-P. Overall, ERG rearrangement was detected in 23.2% (44/190 cases, of which 54.5% (24/44 showed deletion of the 5'end of ERG. PTEN deletion was identified in 10.8% (19/176 cases. Amplification of EGFR and HER2 genes was present in 1.1% (2/178 and 5.8% (10/173 of cases, respectively. Significant correlation between ERG rearrangement and PTEN deletion was identified in this cohort. EGFR and HER2 aberrations occurred more frequently in PCas without ERG rearrangement than in those with ERG rearrangement, although this did not reach statistical significance. Overall, ERG rearrangement was associated with pre-operative PSA values (P = 0.038 and cancer-related death (P = 0.02, but not with the age, clinical T stage, Gleason score, or Ki-67 labeling index (LI. Notably, multivariate analysis including known prognostic markers revealed ERG rearrangement was an independent prognostic factor (P = 0.022. Additionally, ERG rearrangement status was helpful to identify patients with poor prognosis from PCa group with low Ki-67 LI. In summary, we reported that ERG rearrangement was associated with cancer-related death in Chinese PCa patients. Determination of ERG rearrangement status allows stratification of PCa patients into different survival categories.

  16. Gravitation and inertia; a rearrangement of vacuum in gravity

    CERN Document Server

    Ter-Kazarian, Gagik

    2010-01-01

    We address the gravitation and inertia in the framework of 'general gauge principle', which accounts for 'gravitation gauge group' generated by hidden local internal symmetry implemented on the flat space. We connect this group to nonlinear realization of the Lie group of 'distortion' of local internal properties of six-dimensional flat space, which is assumed as a toy model underlying four-dimensional Minkowski space. The agreement between proposed gravitational theory and available observational verifications is satisfactory. We construct relativistic field theory of inertia and derive the relativistic law of inertia. This theory furnishes justification for introduction of the Principle of Equivalence. We address the rearrangement of vacuum state in gravity resulting from these ideas.

  17. Filming the Birth of Molecules and Accompanying Solvent Rearrangement

    DEFF Research Database (Denmark)

    Lee, Jae Hyuk; Wulff, Michael; Bratos, Savo;

    2013-01-01

    Molecules are often born with high energy and large-amplitude vibrations. In solution, a newly formed molecule cools down by transferring energy to the surrounding solvent molecules. The progression of the molecular and solute−solvent cage structure during this fundamental process has been elusive......, and spectroscopic data generally do not provide such structural information. Here, we use picosecond X-ray liquidography (solution scattering) to visualize timedependent structural changes associated with the vibrational relaxation of I2 molecules in two different solvents, CCl4 and cyclohexane. The birth...... and vibrational relaxation of I2 molecules and the associated rearrangement of solvent molecules are mapped out in the form of a temporally varying interatomic distance distribution. The I−I distance increases up to ∼4 Å and returns to the equilibrium distance (2.67 Å) in the ground state, and the first solvation...

  18. SYNTHESIS OF ALLYL PHENYL ETHER AND CLAISEN REARRANGEMENT

    Directory of Open Access Journals (Sweden)

    Gagik Torosyan

    2011-12-01

    Full Text Available It has been established the possibility for phenol allylation on natural zeolites and them analogs. Here is demonstrated the synthesis of allyl phenol, which has wide industrial applications. The offered method in comparison with the traditional methods has more advantages – higher selectivity, smaller material and power resources consumption. It has been obtained the mixture of allylating phenols (30% in general with allyl phenyl ether (1 with 80% yields. At 600 K is obtained allylphenyl ether, at 700 K beginning the formation of allyl phenols, which is the result of direct C-allylation of the aromatic ring. It has been investigated the possibility of Claisen rearrangement in the same conditions. All of that are established by gas-liquid chromatography and liquid chromatography data.

  19. Electronic Structure Rearrangements in Hybrid Ribozyme/Protein Catalysis

    Science.gov (United States)

    Kang, Jiyoung; Kino, Hiori; Field, Martin J.; Tateno, Masaru

    2017-04-01

    We analyzed the electronic structural changes that occur in the reaction cycle of a biological catalyst composed of RNA and protein, and elucidated the dynamical rearrangements of the electronic structure that was obtained from our previous study in which ab initio quantum mechanics/molecular mechanics molecular dynamics simulations were performed. Notable results that we obtained include the generation of a reactive HOMO that is responsible for bond formation in the initial stages of the reaction, and the appearance of a reactive LUMO that is involved in the bond rupture that leads to products. We denote these changes as dynamical induction of the reactive HOMO (DIRH) and LUMO (DIRL), respectively. Interestingly, we also find that the induction of the reactive HOMO is enhanced by the formation of a low-barrier hydrogen bond (LBHB), which, to the best of our knowledge, represents a novel role for LBHBs in enzymatic systems.

  20. Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosomes 3. Evidence for the role of chromosome rearrangement in gene amplification

    Energy Technology Data Exchange (ETDEWEB)

    Stallings, R.L.; Munk, A.C.; Longmire, J.L.; Hildebrand, C.E.; Crawford, B.D.

    1984-12-01

    Cadmium resistant (Cd/sup r/) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cd/sup r/ variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster x mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. The authors speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cd/sup r/ hamster cell lines. 34 references, 3 figures, 1 table.

  1. Most ultraviolet irradiation induced mutations in the nematode Caenorhabditis elegans are chromosomal rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Stewart, H.I.; Rosenbluth, R.E.; Baillie, D.L. (Simon Fraser University, Burnaby, BC (Canada). Department of Biological Sciences, Institute of Molecular Biology)

    1991-07-01

    In this study the utility of 254-nm ultraviolet light (UV) as a magnetic tool in C.elegans is determined. It is demonstrated that irradiation of adult hermaphrodites provides a simple method for the induction of heritable chromosomal rearrangements. A screening protocol was employed that identifies either recessive lethal mutations in the 40 map unit region balanced by the translocation eT1(III;V), or unc-36(III) duplications. Mutations were recovered in 3% of the chromosomes screened after a dose of 120 J/m{sup 2}. This rate resembles that for 1500 R {gamma}-ray-induced mutations selected in a similar manner. The mutations were classified either as lethals (mapping to Linkage Group (LG)III or LGV) or as putative unc-36 duplications. In contrast to the majority of UV-induced mutations analysed in micro-organisms, a large fraction of the C.elegans UV-induced mutations were found to be not simple intragenic lesions, but deficiencies for more than one adjacent gene or more complex events. Preliminary evidence for this conclusion came from the high frequency of mutations that had a dominant effect causing reduced numbers of adult progeny. Subsequently 6 out of 9 analysed LGV mutations were found to be deficiencies. Other specific rearrangements also identified were: one translocation, sT5(II;III), and two unc-36 duplications, sDp8 and sDp9. It was concluded that UV irradiation can easily be used as an additional tool for the analysis of C.elegans chromosomes, and that C.elegans should prove to be a useful organism in which to study the mechanisms whereby UV acts as a mutagen in cells of complex eukaryotes. (author). 46 refs.; 5 figs.; 4 tabs.

  2. The ommatidia of Arca noae: a three-tier structure with a central light-guiding element for the receptor cell.

    Science.gov (United States)

    Roth, Jürgen; Guhl, Bruno; Kloter, Urs; Gehring, Walter J

    2011-07-01

    The compound eyes of ark clams appear to function as an optical system to trigger shell closure against predators. We have analyzed the structure of the ommatidia of Arca noae by thin section electron microscopy and serial sectioning, Concanavalin A-gold labeling and acid phosphatase cytochemistry. Our results demonstrate that the ommatidia are a three-tier structure composed of a central single receptor cell, surrounded and covered by proximal pigment cells followed by rows of distal pigment cells. The receptor cells of Arca noae have no lens and the disks of their receptive segment are derived from sensory cilia. The distal mitochondrial segment in the cytoplasm between the nucleus and the receptive segment is surrounded by a mass of Concanavalin A-reactive glycogen particles. Although both, proximal and distal pigment cells have numerous microvilli, only those of the proximal pigment cells form a well-aligned brush border. The microvilli of the latter are ≈9-11 μm long and have a diameter of ≈70-80 nm. Numerous microlamellar bodies cover them. The microlamellar bodies are stored in acid phosphatase-negative secretory granules of the pigment granule-free apical cytoplasm of proximal pigment cells before their secretion. Observation of living compound eyes indicated that the apex of proximal pigment cells transmitted significantly more light than the surrounding distal pigment cells. Hence, the regular geometry of the brush border seems to be a light-guiding structure for receptor cells similar to an optical fiber.

  3. 32P-incorporation PCR for the detection of rearrangements at the TCR-gamma locus.

    Science.gov (United States)

    Short, M A; Evans, P A; Shiach, C R; Jack, A; Richards, S; Morgan, G J

    1996-03-01

    We have adapted and developed a PCR (polymerase chain reaction)-based technique for the T-cell receptor (TCR)-gamma chain gene, which has subsequently been used for routine diagnosis. Variable-region oligonucleotide primers were chosen from subgroups I and II, and the joining region primer was from the J2 segment. The primers were used to perform a 32P-incorporation PCR, and the products were then separated on an 8% denaturing polyacrylamide gel. In our hands, this technique is more reliable than cold methods, when separation is performed on either agarose or nondenaturing polyacrylamide. The radioactive technique was used to look at 102 T-cell proliferations, of which eight of eight T-acute lymphoblastic leukemia (ALL), 24 of 34 T-non-Hodgkin's leukemia (NHL), and 35 of 60 large granular lymphocyte (LGL) expansions were clonal. Of 122 B-cell proliferations investigated, including 72 cases of B-cell lineage ALL, 36 demonstrated a T-cell rearrangement (33 ALLs and three myelomas). Samples from nonlymphoid tumors were tested and produced a normal distribution ladder of PCR products after autoradiography, a pattern also observed with antenatal and preoperative patients. The radiolabel-incorporation method detected an abnormal pattern of a ladder with prominent dark bands in 29 of 122 B-cell and 27 of 102 T-cell cases and in 0 of 49 of the nonlymphoid and normal samples. The abnormal banding patterns obtained in a proportion of the B- and T-cell cases was not readily discernible by nondenaturing-acrylamide or agarose-separation methods.

  4. Recent applications of the divinylcyclopropane–cycloheptadiene rearrangement in organic synthesis

    Science.gov (United States)

    Krüger, Sebastian

    2014-01-01

    Summary This review summarizes the application of the divinylcyclopropane–cycloheptadiene rearrangement in synthetic organic chemistry. A brief overview of the new mechanistic insights concerning the title reaction is provided as well as a condensed account on the biological relevance of the topic. Heteroatom variants of this rearrangement are covered briefly. PMID:24605138

  5. A Novel Method for Beckmann Rearrangement of Oximes with Silica Sulfuric Acid under Mild Condition

    Institute of Scientific and Technical Information of China (English)

    Lin Fei XIAO; Jia Jian PENG; Chun Gu XIA

    2006-01-01

    Silica sulfuric acid in which sulfuric acid is immobilized on the surface of silica gel via covalent bond has been proved to be green catalyst for liquid-phase Beckmann rearrangement of oximes in dried dioxane at room temperature. Excellent conversion and selectivity were acquired in the Beckmann rearrangement of cyclohexanone oxime. The catalyst system was recycled and reused.

  6. Lecture Capture with Real-Time Rearrangement of Visual Elements: Impact on Student Performance

    Science.gov (United States)

    Yu, P.-T.; Wang, B.-Y.; Su, M.-H.

    2015-01-01

    The primary goal of this study is to create and test a lecture-capture system that can rearrange visual elements while recording is still taking place, in such a way that student performance can be positively influenced. The system we have devised is capable of integrating and rearranging multimedia sources, including learning content, the…

  7. Recent applications of the divinylcyclopropane–cycloheptadiene rearrangement in organic synthesis

    Directory of Open Access Journals (Sweden)

    Sebastian Krüger

    2014-01-01

    Full Text Available This review summarizes the application of the divinylcyclopropane–cycloheptadiene rearrangement in synthetic organic chemistry. A brief overview of the new mechanistic insights concerning the title reaction is provided as well as a condensed account on the biological relevance of the topic. Heteroatom variants of this rearrangement are covered briefly.

  8. A Mild and Highly Efficient Catalyst for Beckmann Rearrangement, BF3·OEt2

    Institute of Scientific and Technical Information of China (English)

    An Na; Pi Hongjun; Liu Lifeng; Du Wenting; Deng Weiping

    2011-01-01

    BF3·OEt2 (Boron trifluoride etherate), an inexpensive and commercially easily available Lewis acid stoichiometrically employed for Beckamann rearrangement in general, was now found to efficiently catalyze Beckmann rearrangement of ketoximes into their corresponding amides (up to 99% yield) in anhydrous acetonitrile under reflux temperature.

  9. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations

    DEFF Research Database (Denmark)

    Rausch, Tobias; Jones, David T W; Zapatka, Marc

    2012-01-01

    of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals...

  10. Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

    NARCIS (Netherlands)

    Kloosterman, W.P.; Hoogstraat, M.; Paling, O.; Tavakoli-Yaraki, M.; Renkens, I.; Vermaat, J.E.; van Roosmalen, M.; van Lieshout, S.; Nijman, I.J.; Roessingh, W.; Van't Slot, R.; van de Belt, J.; Guryev, V.; Koudijs, M.J.; Voest, E.E.; Cuppen, E.

    2011-01-01

    ABSTRACT: BACKGROUND: Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of

  11. Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer

    NARCIS (Netherlands)

    Kloosterman, W.P.; Hoogstraat, M.; Paling, O.; Tavakoli-Yaraki, M.; Renkens, I.; Vermaat, J.S.; Roosmalen, van M.J.; Lieshout, van S.; Nijman, I.J.; Roessingh, W.; Slot, van 't R.; Belt, van de J.

    2011-01-01

    Background - Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. T

  12. The complex photo-rearrangement of a heterocyclic N-oxide : Kinetics from picoseconds to minutes

    NARCIS (Netherlands)

    Cordes, Thorben; Regner, Nadja; Heinz, Björn; Borysova, Elina; Ryseck, Gerald; Gilch, Peter

    2009-01-01

    The photo-induced rearrangement of 2-benzoyl-3-phenylquinoxaline-1,4-dioxide yielding 1,3-dibenzoylbenzimidazolone (DBBI) is studied by time-resolved fluorescence, UV/vis absorption, and IR spectroscopy. This complex rearrangement occurs on time scales ranging from 0.1 ps up to minutes. Processes wi

  13. Regioselective synthesis of heteroaryl triflones by LDA (lithium diisopropylamide)-mediated anionic thia-Fries rearrangement.

    Science.gov (United States)

    Xu, Xiu-Hua; Wang, Xin; Liu, Guo-kai; Tokunaga, Etsuko; Shibata, Norio

    2012-05-18

    Novel heteroaryl triflones including oxindole, pyrazolone, pyridine, and quinoline derivatives have been regioselectively synthesized by LDA-mediated thia-Fries rearrangement for the first time. These reactions are also the first examples of the application of anionic thia-Fries rearrangement in heteroaromatic compounds.

  14. Effective stimulation of growth in MCF-7 human breast cancer cells by inhibition of syntaxin18 by external guide sequence and ribonuclease P.

    Science.gov (United States)

    Bassett, Tyler; Harpur, Brock; Poon, Ho Y; Kuo, Kuo-Hsing; Lee, Chow H

    2008-12-01

    Syntaxin18 (Stx18) is an endoplasmic reticulum (ER)-membrane bound SNARE protein involved in membrane trafficking between the ER and Golgi as well as in phagocytosis. Stx18 has also been shown to physically interact with proteins involved in the cell cycle and apoptosis. These findings suggest the possible role of Stx18 in regulating cell growth. In this study, we used theoretically designed external guide sequence molecule which utilizes RNase P to cleave Stx18 mRNA and down-regulate Stx18 levels in MCF-7 human breast cancer cells. We showed that down-regulation of Stx18 leads to significant enhancement of growth in MCF-7 cells. Consistent with this finding was the observation that over-expression of Stx18 using the CMV promoter led to suppression of cell growth. Over-expressing Stx18 had no effect on c-myc mRNA expression and half-life, suggesting that the mechanism does not involve control at the transcriptional and post-transcriptional level of the c-myc gene. Finally, we showed that Stx18 is over-expressed in clinical human breast cancer. Overall, this study showed that Stx18 plays a role in the growth of human breast cancer cells and provided the basis for further investigation in determining whether it can be used as a prognostic marker and as a molecular target in the treatment of breast cancer.

  15. Correlating structural order with structural rearrangement in dusty plasma liquids: can structural rearrangement be predicted by static structural information?

    Science.gov (United States)

    Su, Yen-Shuo; Liu, Yu-Hsuan; I, Lin

    2012-11-09

    Whether the static microstructural order information is strongly correlated with the subsequent structural rearrangement (SR) and their predicting power for SR are investigated experimentally in the quenched dusty plasma liquid with microheterogeneities. The poor local structural order is found to be a good alarm to identify the soft spot and predict the short term SR. For the site with good structural order, the persistent time for sustaining the structural memory until SR has a large mean value but a broad distribution. The deviation of the local structural order from that averaged over nearest neighbors serves as a good second alarm to further sort out the short time SR sites. It has the similar sorting power to that using the temporal fluctuation of the local structural order over a small time interval.

  16. Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement.

    Science.gov (United States)

    Ohno, Yuko; Ogiyama, Yuki; Kubota, Yoshino; Kubo, Takuya; Ishii, Kojiro

    2016-01-08

    The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.

  17. EWSR1 gene rearrangement occurs in a subset of cutaneous myoepithelial tumors: a study of 18 cases.

    Science.gov (United States)

    Flucke, Uta; Palmedo, Gabriele; Blankenhorn, Nina; Slootweg, Pieter J; Kutzner, Heinz; Mentzel, Thomas

    2011-11-01

    Cutaneous myoepithelial tumors form a clinicopathological spectrum ranging from mixed tumor to myoepithelioma and myoepithelial carcinoma. Recently, EWSR1 rearrangement has been described in a subset of soft tissue myoepithelial tumors, whereas the cutaneous counterparts showed this aberration in a minority of cases. This raises the question whether cutaneous myoepithelial tumors have comparable genetic alterations. We examined 18 cases of cutaneous myoepithelial tumors arising in 7 female and 11 male patients (age range, 34-86 years; mean, 58 years). Eight mixed tumors occurred at the head, and one at the scrotum. Six myoepitheliomas arose at the extremities, and one case each at the back and head. One myoepithelial carcinoma occurred at the cheek. The tumor size ranged from 0.3 to 1.7 cm (mean, 1.0 cm). All mixed tumors and three myoepitheliomas were limited to the dermis. Four myoepitheliomas and the myoepithelial carcinoma involved the subcutis. Mixed tumors and myoepitheliomas were composed of myoepithelial cells with a variable cytomorphology, architecture and stromal background. Ductal structures were seen by definition in mixed tumors. The myoepithelial carcinoma represented an infiltrative dermal neoplasm consisting of atypical spindle cells. Immunohistochemically, all cases tested were positive for EMA and calponin, whereas S100, CK, ASMA and GFAP were expressed in 90%, 80%, 78% and 50% of the cases tested, respectively. By fluorescent in situ hybridization analysis, 7 out of 16 cases (44%) exhibited EWSR1 rearrangement. Four of them were mixed tumors, two were myoepitheliomas and one was a myoepithelial carcinoma, confirming that these lesions represent a spectrum of dermal myoepithelial tumors. Follow-up information, available for five patients (including the patient with a myoepithelial carcinoma), revealed no evidence of disease in all cases (range, 6-72 months). Our study provides a genetic relationship of myoepithelial tumors of the skin with their

  18. Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

    Science.gov (United States)

    Qiu, Zhijun; Zhang, Zhenhua; Roschke, Anna; Varga, Tamas; Aplan, Peter D.

    2017-01-01

    Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression. PMID:28225067

  19. Recurrent SKIL-activating rearrangements in ETS-negative prostate cancer.

    Science.gov (United States)

    Annala, Matti; Kivinummi, Kati; Tuominen, Joonas; Karakurt, Serdar; Granberg, Kirsi; Latonen, Leena; Ylipää, Antti; Sjöblom, Liisa; Ruusuvuori, Pekka; Saramäki, Outi; Kaukoniemi, Kirsi M; Yli-Harja, Olli; Vessella, Robert L; Tammela, Teuvo L J; Zhang, Wei; Visakorpi, Tapio; Nykter, Matti

    2015-03-20

    Prostate cancer is the third most common cause of male cancer death in developed countries, and one of the most comprehensively characterized human cancers. Roughly 60% of prostate cancers harbor gene fusions that juxtapose ETS-family transcription factors with androgen regulated promoters. A second subtype, characterized by SPINK1 overexpression, accounts for 15% of prostate cancers. Here we report the discovery of a new prostate cancer subtype characterized by rearrangements juxtaposing the SMAD inhibitor SKIL with androgen regulated promoters, leading to increased SKIL expression. SKIL fusions were found in 6 of 540 (1.1%) prostate cancers and 1 of 27 (3.7%) cell lines and xenografts. 6 of 7 SKIL-positive cancers were negative for ETS overexpression, suggesting mutual exclusivity with ETS fusions. SKIL knockdown led to growth arrest in PC-3 and LNCaP cell line models of prostate cancer, and its overexpression led to increased invasiveness in RWPE-1 cells. The role of SKIL as a prostate cancer oncogene lends support to recent studies on the role of TGF-β signaling as a rate-limiting step in prostate cancer progression. Our findings highlight SKIL as an oncogene and potential therapeutic target in 1-2% of prostate cancers, amounting to an estimated 10,000 cancer diagnoses per year worldwide.

  20. In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies.

    Science.gov (United States)

    McDermott, Martina; Eustace, Alex J; Busschots, Steven; Breen, Laura; Crown, John; Clynes, Martin; O'Donovan, Norma; Stordal, Britta

    2014-01-01

    The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical

  1. In vitro development of chemotherapy and targeted therapy drug-resistant cancer cell lines: A practical guide with case studies

    Directory of Open Access Journals (Sweden)

    Martina eMcDermott

    2014-03-01

    Full Text Available The development of a drug-resistant cell line can take from 3-18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval and optimising the dose of drug for the parent cell line. Clinically-relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between 2-8 fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically-relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299, H460 and temozolomide-resistant melanoma (Malme-3M and HT144 cell lines. Continuous selection produced lapatinib-resistant breast cancer cell line (HCC1954. Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug

  2. Dienone-phenol Rearrangement of C-9 Oxygenated Decalinic Dienone and Analogs through B-Ring Cleavage

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Dehydrogenation of 9-hydroxy decalinic enones and analogs with DDQ resulted in a formal dienone-phenol type rearrangement via B-ring cleavage, while the corresponding dienone acetates underwent base-catalyzed formal dienone-phenol type rearrangement analogously.

  3. Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies.

    NARCIS (Netherlands)

    Vissers, L.E.L.M.; Stankiewicz, P.; Yatsenko, S.A.; Crawford, E.; Creswick, H.; Proud, V.K.; Vries, B. de; Pfundt, R.; Marcelis, C.L.M.; Zackowski, J.; Bi, W.; Geurts van Kessel, A.H.M.; Lupski, J.R.; Veltman, J.A.

    2007-01-01

    Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. H

  4. Study of particle rearrangement, compression behavior and dissolution properties after melt dispersion of ibuprofen, Avicel and Aerosil

    OpenAIRE

    Mallick, Subrata; Kumar Pradhan, Saroj; Chandran, Muronia; Acharya, Manoj; Digdarsini, Tanmayee; Mohapatra, Rajaram

    2011-01-01

    Particle rearrangements, compaction under pressure and in vitro dissolution have been evaluated after melt dispersion of ibuprofen, Avicel and Aerosil. The Cooper–Eaton and Kuno equations were utilized for the determination of particle rearrangement and compression behavior from tap density and compact data. Particle rearrangement could be divided into two stages as primary and secondary rearrangement. Transitional tapping between the stages was found to be 20–25 taps in ibuprofen crystalline...

  5. Subtelomeric chromosomal rearrangements in a large cohort of unexplained intellectually disabled individuals in Indonesia: A clinical and molecular study

    NARCIS (Netherlands)

    Mundhofir, F.E.P.; Nillesen, W.M.; Bon, B.W.M. van; Smeets, D.; Pfundt, R.P.; Ven-Schobers, G. van de; Ruiterkamp-Versteeg, M.; Winarni, T.I.; Hamel, B.C.J.; Yntema, H.G.; Faradz, S.M.H.

    2013-01-01

    CONTEXT: Unbalanced subtelomeric chromosomal rearrangements are often associated with intellectual disability (ID) and malformation syndromes. The prevalence of such rearrangements has been reported to be 5-9% in ID populations. AIMS: To study the prevalence of subtelomeric rearrangements in the Ind

  6. NotI jumping and linking clones as a tool for genome mapping and analysis of chromosome rearrangements in different tumors.

    Science.gov (United States)

    Zabarovsky, E R; Kashuba, V I; Gizatullin, R Z; Winberg, G; Zabarovska, V I; Erlandsson, R; Domninsky, D A; Bannikov, V M; Pokrovskaya, E; Kholodnyuk, I; Petrov, N; Zakharyev, V M; Kisselev, L L; Klein, G

    1996-01-01

    Long-range restriction site maps are of central importance for mapping the human genome. The use of clones from linking and jumping libraries for genome mapping offers a promising alternative to the laborious procedures used up until now. In the present review, this research field is analyzed with particular emphasis on the implementation of a shot-gun sequencing strategy for genome mapping and the use of NotI linking clones for analysis of rearrangements in tumors and tumor cell lines.

  7. Genomic characterization of large rearrangements of the LDLR gene in Czech patients with familial hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Fajkus Jiří

    2010-07-01

    Full Text Available Abstract Background Mutations in the LDLR gene are the most frequent cause of Familial hypercholesterolemia, an autosomal dominant disease characterised by elevated concentrations of LDL in blood plasma. In many populations, large genomic rearrangements account for approximately 10% of mutations in the LDLR gene. Methods DNA diagnostics of large genomic rearrangements was based on Multiple Ligation dependent Probe Amplification (MLPA. Subsequent analyses of deletion and duplication breakpoints were performed using long-range PCR, PCR, and DNA sequencing. Results In set of 1441 unrelated FH patients, large genomic rearrangements were found in 37 probands. Eight different types of rearrangements were detected, from them 6 types were novel, not described so far. In all rearrangements, we characterized their exact extent and breakpoint sequences. Conclusions Sequence analysis of deletion and duplication breakpoints indicates that intrachromatid non-allelic homologous recombination (NAHR between Alu elements is involved in 6 events, while a non-homologous end joining (NHEJ is implicated in 2 rearrangements. Our study thus describes for the first time NHEJ as a mechanism involved in genomic rearrangements in the LDLR gene.

  8. Genomic Rearrangement in Endogenous Long Terminal Repeat Retrotransposons of Rice Lines Introgressed by Wild Rice (Zizania latifolia Griseb.)

    Institute of Scientific and Technical Information of China (English)

    Ye SHEN; Xiu-Yun LIN; Xiao-Hui SHAN; Chun-Jing LIN; Fang-Pu HAN; Jin-Song PANG; Bao LIU

    2005-01-01

    Stochastic introgression of alien DNA may impose a genomic stress to the recipient genome.Herein, we report that apparent de novo genomic rearrangements in 10 of 13 selected endogenous, low-copy, and potentially active long terminal repeat (LTR) retrotransposons occurred in one or more of threerice lines studied that were introgressed by wild rice (Zizania latifolia Griseb.). For nine retrotransposons inwhich both the reverse-transcriptase (RT) region and the LTR region were available, largely concordantrearrangements occurred at both regions in five elements and at the RT region only in the remaining fourelements. A marked proportion of the genomic changes was shared by two or all three introgression linesthat were derived from a single F1 plant. This indicates that most of the genomic changes occurred at earlydevelopmental stages of the F1 somatic cells, which then gave rise to germline cells, and, hence, ensuredinheritance of the changes to later generations. Possible causes and potential implications of the introgres-sion-induced genomic rearrangements in LTR retrotransposons are discussed in the context of plant ge-nome evolution and breeding.

  9. Protein expression guided chemical profiling of living cells by the simultaneous observation of Raman scattering and anti-Stokes fluorescence emission

    Science.gov (United States)

    Chiu, Liang-da; Ichimura, Taro; Sekiya, Takumasa; Machiyama, Hiroaki; Watanabe, Tomonobu; Fujita, Hideaki; Ozawa, Takeaki; Fujita, Katsumasa

    2017-01-01

    Our current understanding of molecular biology provides a clear picture of how the genome, transcriptome and proteome regulate each other, but how the chemical environment of the cell plays a role in cellular regulation remains much to be studied. Here we show an imaging method using hybrid fluorescence-Raman microscopy that measures the chemical micro-environment associated with protein expression patterns in a living cell. Simultaneous detection of fluorescence and Raman signals, realised by spectrally separating the two modes through the single photon anti-Stokes fluorescence emission of fluorescent proteins, enables the accurate correlation of the chemical fingerprint of a specimen to its physiological state. Subsequent experiments revealed the slight chemical differences that enabled the chemical profiling of mouse embryonic stem cells with and without Oct4 expression. Furthermore, using the fluorescent probe as localisation guide, we successfully analysed the detailed chemical content of cell nucleus and Golgi body. The technique can be further applied to a wide range of biomedical studies for the better understanding of chemical events during biological processes. PMID:28272392

  10. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Science.gov (United States)

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  11. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Weiming Wang

    Full Text Available CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN or Transcription activator-like effector nuclease (TALEN. In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9 and single guided RNAs (sgRNAs. We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  12. Multiple forms of atypical rearrangements generating supernumerary derivative chromosome 15

    Directory of Open Access Journals (Sweden)

    Sigman Marian

    2008-01-01

    Full Text Available Abstract Background Maternally-derived duplications that include the imprinted region on the proximal long arm of chromosome 15 underlie a complex neurobehavioral disorder characterized by cognitive impairment, seizures and a substantial risk for autism spectrum disorders1. The duplications most often take the form of a supernumerary pseudodicentric derivative chromosome 15 [der(15] that has been called inverted duplication 15 or isodicentric 15 [idic(15], although interstitial rearrangements also occur. Similar to the deletions found in most cases of Angelman and Prader Willi syndrome, the duplications appear to be mediated by unequal homologous recombination involving low copy repeats (LCR that are found clustered in the region. Five recurrent breakpoints have been described in most cases of segmental aneuploidy of chromosome 15q11-q13 and previous studies have shown that most idic(15 chromosomes arise through BP3:BP3 or BP4:BP5 recombination events. Results Here we describe four duplication chromosomes that show evidence of atypical recombination events that involve regions outside the common breakpoints. Additionally, in one patient with a mosaic complex der(15, we examined homologous pairing of chromosome 15q11-q13 alleles by FISH in a region of frontal cortex, which identified mosaicism in this tissue and also demonstrated pairing of the signals from the der(15 and the normal homologues. Conclusion Involvement of atypical BP in the generation of idic(15 chromosomes can lead to considerable structural heterogeneity.

  13. Structural rearrangement through lanthanide contraction in dinuclear complexes.

    Science.gov (United States)

    Hutchings, Amy-Jayne; Habib, Fatemah; Holmberg, Rebecca J; Korobkov, Ilia; Murugesu, Muralee

    2014-02-17

    A new series of lanthanide complexes was synthesized, and the geometry and preliminary magnetic measurements of the complexes were explored. The specific ligand used (N'-(2-hydroxy-3-methoxybenzylidene)benzhydrazide) (H2hmb) was synthesized using a Schiff-base approach and was employed due to the presence of a coordination pocket that is able to accommodate magnetically selective lanthanide ions. The series can be divided into two groups that are categorized by a drastic structural rearrangement. The first group, Type I, contains six analogous complexes with the formula [M(III)2(Hhmb)3(NCS)3]·2MeOH·py (M = Y 1, Eu 2, Gd 3, Tb 4, Dy 5, Ho 6), while the second group, Type II, contains two dinuclear complexes with formula [M(III)2(Hhmb)2(NCS)4(MeOH)2] (M = Er 7, and Yb 8). Single-crystal X-ray analysis revealed that all M(III) ions in Type I exhibit monocapped distorted square antiprismatic geometries, while those of Type II exhibit distorted dodecahedron geometry. The direct current and alternating current magnetic measurements were carried out on all complexes, with 5, 7, and 8 exhibiting slow relaxation of the magnetization under an applied optimum dc field. Furthermore, complex 8 is the first example of a dinuclear Yb-based single-molecule magnet showing field-dependent multiple relaxation processes.

  14. Ferrier rearrangement promoted by an electrochemically generated zirconium catalyst.

    Science.gov (United States)

    Stevanović, Dragana; Pejović, Anka; Damljanović, Ivan; Minić, Aleksandra; Bogdanović, Goran A; Vukićević, Mirjana; Radulović, Niko S; Vukićević, Rastko D

    2015-04-30

    In situ generated zirconium catalyst from a sacrificial zirconium anode was successfully applied to promote Ferrier rearrangement of 3,4,5-tri-O-acetyl-D-glucal and 6-deoxy-3,4-di-O-acetyl-L-glucal (3,4-di-O-acetyl-L-rhamnal) in the presence of three thiols and eleven thiophenols as nucleophiles. A simple constant current electrolysis (20 mA, 0.4 F mol(-1)) of an acetonitrile solution of lithium perchlorate (0.1 M) containing the corresponding glycal and S-nucleophiles, using a zirconium anode and a platinum cathode resulted in the successful synthesis of the corresponding 2,3-unsaturated peracetylated thioglycosides (with an average anomer ratio α/β=4.129 in the case of peracetylated D-glucal and 8.740 in the case of L-rhamnal). The same procedure proved to be appropriate in synthesizing dihydropyran derivatives ('C-glycosides') using allyltrimethylsilane as the nucleophile (only 'α-anomers' were obtained). All new compounds were fully characterized by spectral data, whereas single-crystal X-ray analysis was performed for two thioglycosides.

  15. On the Complexity of Rearrangement Problems under the Breakpoint Distance

    CERN Document Server

    Kovac, Jakub

    2011-01-01

    Tannier et al. introduced a generalization of breakpoint distance for multichromosomal genomes. They showed that the median problem under the breakpoint distance is solvable in polynomial time in the multichromosomal circular and mixed models. This is intriguing, since in all other rearrangement models (DCJ, reversal, unichromosomal or multilinear breakpoint models), the problem is NP-hard. The complexity of the small or even the large phylogeny problem under the breakpoint distance remained an open problem. We improve the algorithm for the median problem and show that it is equivalent to the problem of finding maximum cardinality non-bipartite matching (under linear reduction). On the other hand, we prove that the more general small phylogeny problem is NP-hard. Surprisingly, we show that it is already NP-hard (or even APX-hard) for 4 species (a quartet phylogeny). In other words, while finding an ancestor for 3 species is easy, already finding two ancestors for 4 species is hard. We also show that, in the u...

  16. Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti.

    Science.gov (United States)

    Yokota, Keisuke; Fukai, Eigo; Madsen, Lene H; Jurkiewicz, Anna; Rueda, Paloma; Radutoiu, Simona; Held, Mark; Hossain, Md Shakhawat; Szczyglowski, Krzysztof; Morieri, Giulia; Oldroyd, Giles E D; Downie, J Allan; Nielsen, Mette W; Rusek, Anna Maria; Sato, Shusei; Tabata, Satoshi; James, Euan K; Oyaizu, Hiroshi; Sandal, Niels; Stougaard, Jens

    2009-01-01

    Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

  17. Abnormal meiotic recombination with complex chromosomal rearrangement in an azoospermic man.

    Science.gov (United States)

    Wang, Liu; Iqbal, Furhan; Li, Guangyuan; Jiang, Xiaohua; Bukhari, Ihtisham; Jiang, Hanwei; Yang, Qingling; Zhong, Liangwen; Zhang, Yuanwei; Hua, Juan; Cooke, Howard J; Shi, Qinghua

    2015-06-01

    Spermatocyte spreading and immunostaining were applied to detect meiotic prophase I progression, homologous chromosome pairing, synapsis and recombination in an azoospermic reciprocal translocation 46, XY, t(5;7;9;13)(5q11;7p11;7p15;9q12;13p12) carrier. Histological examination of the haematoxylin and eosin stained testicular sections revealed reduced germ cells with no spermatids or sperm in the patient. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay showed apoptotic cells in testicular sections of translocation carrier. Immnunofluorescence analysis indicated the presence of an octavalent in all the pachytene spermatocytes analysed in the patient. Meiotic progression was disturbed, as an increase in zygotene (P recombination frequency was observed on 5p, 5q, 7q, 9p and 13q in the translocation carrier compared with the reported controls. A significant reduction in XY recombination frequency was also found in the participants. Our results indicated that complex chromosomal rearrangements can impair synaptic integrity of translocated chromosomes, which may reduce chromosomal recombination on translocated as well as non-translocated chromosomes, a phenomenon commonly known as interchromosomal effect.

  18. Postsplenectomy cytomegaloviral mononucleosis: marked lymphocytosis, TCRgamma gene rearrangements, and impaired IgM response.

    Science.gov (United States)

    Han, Xiang Y; Lin, Pei; Amin, Hesham M; Ferrajoli, Alessandra

    2005-04-01

    People who have undergone splenectomy mount a poor IgM response to bacterial polysaccharide vaccines. Whether this defect is true during natural bacterial and viral infections is unknown. We present 2 cases of postsplenectomy cytomegalovirus (CMV)-induced mononucleosis with impaired IgM but normal to augmented IgG response. The cases presented initial diagnostic challenges owing to a prolonged course of infection, marked lymphocytosis (peak lymphocyte count, 27,900/microL [27.9 10(9)/L]), clonal T-cell proliferation with T-cell receptor g gene rearrangements, and remote history of splenectomy. However, the acute nature of the infections, serial determinations of the anti-CMV IgM and IgG, exclusion of other causes, and detection of CMV in the blood established the diagnosis and revealed the deranged antibody response. The infections resolved without specific treatment. These cases suggest that the spleen might be a primary site for specific anti-CMV IgM response.

  19. Detection of immunoglobulin IGH gene rearrangements on formalin-fixed, paraffin embedded tissue in lymphoid malignancies.

    Science.gov (United States)

    Moharrami, G; Ghorbian, S; Seifi, M; Estiar, M A; Fakhrjoo, A; Sakhinia, M; Sakhinia, E

    2014-01-01

    Human lymphomas are aggressive malignant diseases, which can be categorized based on their B and T cell lineage. B-cell lymphomas form around 90% of the total lymphoma cases, the remnants of malignancies arise from the T cell branch. Lymphomas are mostly characterized as clonal proliferations of specific tumor cells. The detection of malignant lymphomas are extensively investigated by their morphological features, immunohistochemistry and flowcytometric immunophenotyping, but in some of cases remained unknown. The BIOMED-2 protocols were used to determine the clonality of IGH gene rearrangements in patients with lymphoma. PCR amplification was performed on FFPE of 50 patients with B-cell lymphoma, which consisted of 11 cases with HLs, 25 cases of B-NHLs and 14 cases of B-LPD (lymphoproliferative disorders) that diagnosed as unclassifiable lymphoma. The rate of positive clonality was detected in 96% (24/25) of B-NHLs, whereas in 4% (1/25) of cases clonality was showed in a polyclonal pattern. In B-HLs, 82% (9/11) of cases showed clonality and 18% (2/11) of the cases showed polyclonality. The rate of positive clonality observed in 64.3% (9/14) of cases with B-LPD and 35.7% (5/14) of cases clonality was not detected in any of immunoglobulin gene family (FR1, FR2, FR3). In groups with DLBCL, clonality was detected in 95% (19/20) of the cases. In patients diagnosed with FL and MALTs 100% cases showed clonality for complete IGH. Our study revealed that EuroClonality BIOMED-2 protocols could be considered as a valuable and reliable method for clonality detection, especially in IGH analysis.

  20. The Importance of an In-depth Study of Immunoglobulin Gene Rearrangements When Ascertaining the Clonal Relationship between Concomitant Chronic Lymphocytic Leukemia and Multiple Myeloma

    Science.gov (United States)

    Trudel, Stéphanie; Ghamlouch, Hussein; Dremaux, Julie; Delette, Caroline; Harrivel, Véronique; Marolleau, Jean-Pierre; Gubler, Brigitte

    2016-01-01

    Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers [particularly solid tumors (3–5)] can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare [for a review of the few reported cases, see Ref. (6)]. The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing, and somatic mutations—events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements [clonality assessment, V(D)J sequencing, and somatic hypermutation analysis] in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible

  1. The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

    Science.gov (United States)

    Ardini, Elena; Bosotti, Roberta; Borgia, Andrea Lombardi; De Ponti, Cristina; Somaschini, Alessio; Cammarota, Rosaria; Amboldi, Nadia; Raddrizzani, Laura; Milani, Andrea; Magnaghi, Paola; Ballinari, Dario; Casero, Daniele; Gasparri, Fabio; Banfi, Patrizia; Avanzi, Nilla; Saccardo, Maria B; Alzani, Rachele; Bandiera, Tiziano; Felder, Eduard; Donati, Daniele; Pesenti, Enrico; Sartore-Bianchi, Andrea; Gambacorta, Marcello; Pierotti, Marco A; Siena, Salvatore; Veronese, Silvio; Galvani, Arturo; Isacchi, Antonella

    2014-12-01

    The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

  2. Guided bone regeneration in pig calvarial bone defects using autologous mesenchymal stem/progenitor cells - a comparison of different tissue sources.

    Science.gov (United States)

    Stockmann, Philipp; Park, Jung; von Wilmowsky, Cornelius; Nkenke, Emeka; Felszeghy, Endre; Dehner, Jan-Friedrich; Schmitt, Christian; Tudor, Christian; Schlegel, Karl Andreas

    2012-06-01

    Due to donor side morbidity and the absence of osteogenic properties in bone substitutes, there is a growing need for an alternative to traditional bone grafting within the scope of tissue engineering. This animal study was conducted to compare the in vivo osteogenic potential of adipose-derived (AD), periosteum-derived (PD) and bone marrow-derived (BM) mesenchymal stem/progenitor cells (MSC). Autologous mesenchymal stem/progenitor cells of named tissue origin were induced into osteogenic differentiation following in vitro cell expansion. Ex vivo cultivated cells were seeded on a collagen scaffold and subsequently added to freshly created monocortical calvarial bone defects in 21 domestic pigs. Pure collagen scaffold served as a control defect. The animals were sacrificed at specific time points and de novo bone formation was quantitatively analyzed by histomorphometry. Bone volume/total defect volume (BV/TV) and the mineralization rate of newly formed bone were compared among the groups. In the early stages of wound healing, up to 30 days, the test defects did not show better bone regeneration than those in the control defect, but the bone healing process in the test defects was accelerated in the later stage compared to those in the control defect. All the test defects showed complete osseous healing after 90 days compared to those in the control defect. During the observation period, no significant differences in BV/TV and mineralization of newly formed bone among the test defects were observed. Irrespective of the tissue sources of MSC, the speed and pattern of osseous healing after cell transplantations into monocortical bone defects were comparable. Our results indicate that the efficiency of autologous AD-MSC, PD-MSC and BM-MSC transplantation following ex vivo cell expansion is not significantly different for the guided regeneration of bone defects.

  3. Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Manchineella, Shivaprasad; Thrivikraman, Greeshma; Basu, Bikramjit; Govindaraju, T

    2016-09-07

    Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications.

  4. Effect of drying history on swelling properties and cell attachment to oligo(poly(ethylene glycol) fumarate) hydrogels for guided tissue regeneration applications.

    Science.gov (United States)

    Temenoff, Johnna S; Steinbis, Emily S; Mikos, Antonios G

    2003-01-01

    In these experiments, the effects of the drying history of hydrogels made from a novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF) with two different poly(ethylene glycol) (PEG) molecular weights (approximately 920 (1K) and 9110 (10K) g/mol), were investigated. The hydrogels were either formed, dried and then swelled, representing what may occur in the case of a pre-formed membrane for guided tissue regeneration, or were formed and swelled immediately, as may occur with an injectable material for such applications. Subsequently, swelling properties, sol fraction and polymer network structure (as indicated by differential scanning calorimetry), as well as attachment of human dermal fibroblasts to these hydrogels at 4 and 24 h was examined. It was found that drying before swelling caused a significant reduction in final fold swelling of OPF hydrogels, regardless of OPF formulation or method of drying (air-dried or vacuum-dried) (e.g. PEG 10K swollen first: 13.94 +/- 0.35 vs. vacuum first: 6.53 +/- 0.12; PEG 1K swollen first: 8.99 +/- 0.47 vs. vacuum first: 2.26 +/- 0.08). This decreased swelling correlated to significantly higher cell attachment (% seeded) to these hydrogels at 24 h (PEG 10K vacuum first: 21.1 +/- 4.7% vs. swollen first: 7.1 +/- 5.5%; PEG 1K vacuum first: 58.2 +/- 2% vs. swollen first: 7.4 +/- 2.2%). LIVE/DEAD staining followed by microscopic analysis revealed attached cells were viable, yet rounded, and that, in the case of the PEG 1K dried-first samples, undulations in the surface visible in the hydrated state may have affected cell adhesion. Regardless of treatment, all hydrogels showed significantly less cell attachment than the tissue culture polystyrene control after 24 h (104.9 +/- 4.4%). These results suggest that, by altering the PEG molecular weight used in synthesis, OPF hydrogels may be tailored to produce desired swelling properties and reduce non-specific cell adhesion for either injectable or pre-formed applications, thus

  5. Using C-arm x-ray imaging to guide local reporter probe delivery for tracking stem cell engraftment.

    Science.gov (United States)

    Kedziorek, Dorota A; Solaiyappan, Meiyappan; Walczak, Piotr; Ehtiati, Tina; Fu, Yingli; Bulte, Jeff W M; Shea, Steven M; Brost, Alexander; Wacker, Frank K; Kraitchman, Dara L

    2013-01-01

    Poor cell survival and difficulties with visualization of cell delivery are major problems with current cell transplantation methods. To protect cells from early destruction, microencapsulation methods have been developed. The addition of a contrast agent to the microcapsule also could enable tracking by MR, ultrasound, and X-ray imaging. However, determining the cell viability within the microcapsule still remains an issue. Reporter gene imaging provides a way to determine cell viability, but delivery of the reporter probe by systemic injection may be hindered in ischemic diseases. In the present study, mesenchymal stem cells (MSCs) were transfected with triple fusion reporter gene containing red fluorescent protein, truncated thymidine kinase (SPECT/PET reporter) and firefly luciferase (bioluminescence reporter). Transfected cells were microencapsulated in either unlabeled or perfluorooctylbromide (PFOB) impregnated alginate. The addition of PFOB provided radiopacity to enable visualization of the microcapsules by X-ray imaging. Before intramuscular transplantation in rabbit thigh muscle, the microcapsules were incubated with D-luciferin, and bioluminescence imaging (BLI) was performed immediately. Twenty-four and forty-eight hours post transplantation, c-arm CT was used to target the luciferin to the X-ray-visible microcapsules for BLI cell viability assessment, rather than systemic reporter probe injections. Not only was the bioluminescent signal emission from the PFOB-encapsulated MSCs confirmed as compared to non-encapsulated, naked MSCs, but over 90% of injection sites of PFOB-encapsulated MSCs were visible on c-arm CT. The latter aided in successful targeting of the reporter probe to injection sites using conventional X-ray imaging to determine cell viability at 1-2 days post transplantation. Blind luciferin injections to the approximate location of unlabeled microcapsules resulted in successful BLI signal detection in only 18% of injections. In conclusion

  6. Synchronized aromaticity as an enthalpic driving force for the aromatic Cope rearrangement.

    Science.gov (United States)

    Babinski, David J; Bao, Xiaoguang; El Arba, Marie; Chen, Bo; Hrovat, David A; Borden, Weston Thatcher; Frantz, Doug E

    2012-10-03

    We report herein experimental and theoretical evidence for an aromatic Cope rearrangement. Along with several successful examples, our data include the first isolation and full characterization of the putative intermediate that is formed immediately after the initial [3,3] sigmatropic rearrangement. Calculations at the B3LYP/6-31G(d) level of theory predict reaction energy barriers in the range 22-23 kcal/mol for the [3,3]-rearrangement consistent with the exceptionally mild reaction conditions for these reactions. The experimental and computational results support a significant enthalpic contribution of the concomitant pyrazole ring formation that serves as both a kinetic and thermodynamic driving force for the aromatic Cope rearrangement.

  7. Unexpected Stereospecific Rearrangement-Addition Reaction of Trisubstituted Gibberellin Epoxides with Trimethylaluminium

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A novel rearrangement-addition reaction of trisubstituted gibberellin epoxides with trimethylaluminium is reported. The reaction proceeds stereospecifically to give tertiary methyl alcohols. The possible mechanism for the reaction is also discussed.

  8. Maternal acute lymphoctic leukemia with rearrangement of the mixed lineage leukemia gene occurring during pregnancy.

    Science.gov (United States)

    Aljurf, Mahmoud; Nassar, Amr; Saleh, Abu J; Almhareb, Fahed; Alzahrani, Hazzaa; Walter, Claudia; Bakr, Mohammad; Ahmed, Syed Osman; Chaudhri, Naeem

    2009-01-01

    Acute lymphoblastic leukemia (ALL) is a relatively rare disease during pregnancy, accounting for about 15% of all cases of pregnancy-associated leukemia. Although mixed lineage leukemia gene (MLL) rearrangement is the dominant genetic aberration in infantile acute leukemia, the occurrence of MLL gene rearrangement in maternal ALL occurring during pregnancy has not been reported. Out of 31 cases of maternal leukemia diagnosed during pregnancy at our institution, 5 were ALL cases. Three of the 5 patients had MLL gene rearrangement. The data for these 5 patients are presented in this report. We believe that the association of MLL gene rearrangement with maternal leukemia is biologically plausible and this observation needs to be validated in a larger cohort of pregnancy-associated maternal leukemia cases.

  9. Low frequency of large genomic rearrangements of BRCA1 and BRCA2 in western Denmark

    DEFF Research Database (Denmark)

    Thomassen, Mads; Gerdes, Anne-Marie; Cruger, Dorthe;

    2006-01-01

    Germline mutations in BRCA1 and BRCA2 predispose female carriers to breast and ovarian cancer. The majority of mutations identified are small deletions or insertions or are nonsense mutations. Large genomic rearrangements in BRCA1 are found with varying frequencies in different populations......, but BRCA2 rearrangements have not been investigated thoroughly. The objective in this study was to determine the frequency of large genomic rearrangements in BRCA1 and BRCA2 in a large group of Danish families with increased risk of breast and ovarian cancer. A total of 617 families previously tested...... negative for mutations involving few bases were screened with multiplex ligation-dependent probe amplification (MLPA). Two deletions in BRCA1 were identified in three families; no large rearrangements were detected in BRCA2. The large deletions constitute 3.8% of the BRCA1 mutations identified, which...

  10. Frequent mitochondrial gene rearrangements at the hymenopteran nad3-nad5 junction.

    Science.gov (United States)

    Dowton, Mark; Castro, Lyda R; Campbell, Sarah L; Bargon, Sharmilla D; Austin, Andrew D

    2003-05-01

    We characterized the organization of mitochondrial genes from a diverse range of hymenopterans. Of the 21 taxa characterized, 12 had distinct, derived organizations. Some rearrangements were consistent with the duplication-random loss mechanism, while others were not. Local inversions were relatively common, i.e., rearrangements characterized by the movement of genes from one mitochondrial strand to the other, opposite or close to their ancestral position. This type of rearrangement is inconsistent with the duplication/random loss model of mitochondrial gene rearrangement. Instead, they are best explained by the operation of recombination. Taxa with derived organizations were restricted to a single, monophyletic group of wasps, the Apocrita, which comprise about 90% of all hymenopterans.

  11. Mycoreovirus genome alterations: similarities to and differences from rearrangements reported for other reoviruses.

    Science.gov (United States)

    Tanaka, Toru; Eusebio-Cope, Ana; Sun, Liying; Suzuki, Nobuhiro

    2012-01-01

    The family Reoviridae is one of the largest virus families with genomes composed of 9-12 double-stranded RNA segments. It includes members infecting organisms from protists to humans. It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. Recently distinct genetic alterations were reported for members of the genus Mycoreovirus, Mycoreovirus 1 (MyRV1), and MyRV3 with 11 (S1-S11) and 12 genome segments (S1-S12), respectively. While MyRV3 S8 is lost during subculturing of infected host fungal strains, MyRV1 rearrangements undergo alterations spontaneously and inducibly. The inducible MyRV1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus Cryphonectria parasitica hypovirus 1 (CHV1). A total of 5 MyRV1 variants with genome rearranged segments (S1-S3, S6 and S10) are generated in the background of a single viral strain in the presence of CHV1 p29 supplied either transgenically or by coinfection. MyRV1 S4 and S10 are rearranged, albeit very infrequently, in a CHV1 p29 independent fashion. A variant of MyRV1 with substantial deletions in both S4 and S10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type MyRV1. In vivo and in vitro interactions of CHV1 p29 and MyRV1 VP9 are implicated in the induction of MyRV1 rearrangements. However, the mechanism underlying p29-mediated rearrangements remains largely unknown. MyRV1 S4 rearrangements spontaneously occurred independently of CHV1 p29. In the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these MyRV1 and MyRV3 variants contribute to functional analyses of the protein products encoded by those rearranged segments.

  12. Evolving gene regulatory networks into cellular networks guiding adaptive behavior: an outline how single cells could have evolved into a centralized neurosensory system.

    Science.gov (United States)

    Fritzsch, Bernd; Jahan, Israt; Pan, Ning; Elliott, Karen L

    2015-01-01

    Understanding the evolution of the neurosensory system of man, able to reflect on its own origin, is one of the major goals of comparative neurobiology. Details of the origin of neurosensory cells, their aggregation into central nervous systems and associated sensory organs and their localized patterning leading to remarkably different cell types aggregated into variably sized parts of the central nervous system have begun to emerge. Insights at the cellular and molecular level have begun to shed some light on the evolution of neurosensory cells, partially covered in this review. Molecular evidence suggests that high mobility group (HMG) proteins of pre-metazoans evolved into the definitive Sox [SRY (sex determining region Y)-box] genes used for neurosensory precursor specification in metazoans. Likewise, pre-metazoan basic helix-loop-helix (bHLH) genes evolved in metazoans into the group A bHLH genes dedicated to neurosensory differentiation in bilaterians. Available evidence suggests that the Sox and bHLH genes evolved a cross-regulatory network able to synchronize expansion of precursor populations and their subsequent differentiation into novel parts of the brain or sensory organs. Molecular evidence suggests metazoans evolved patterning gene networks early, which were not dedicated to neuronal development. Only later in evolution were these patterning gene networks tied into the increasing complexity of diffusible factors, many of which were already present in pre-metazoans, to drive local patterning events. It appears that the evolving molecular basis of neurosensory cell development may have led, in interaction with differentially expressed patterning genes, to local network modifications guiding unique specializations of neurosensory cells into sensory organs and various areas of the central nervous system.

  13. Synthesis of Benzofuran Derivatives via Rearrangement and Their Inhibitory Activity on Acetylcholinesterase

    Directory of Open Access Journals (Sweden)

    Ling-Yi Kong

    2010-11-01

    Full Text Available During a synthesis of coumarins to obtain new candidates for treating Alzheimer’s Disease (AD, an unusual rearrangement of a benzopyran group to a benzofuran group occurred, offering a novel synthesis pathway of these benzofuran derivatives. The possible mechanism of the novel rearrangement was also discussed. All of the benzofuran derivatives have weak anti-AChE activities compared with the reference compound, donepezil.

  14. Hominoid chromosomal rearrangements on 17q map to complex regions of segmental duplication

    OpenAIRE

    Cardone, Maria Francesca; Jiang, Zhaoshi; D'Addabbo, Pietro; Archidiacono, Nicoletta; Rocchi, Mariano; Eichler, Evan E.; Ventura, Mario

    2008-01-01

    Background Chromosomal rearrangements, such as translocations and inversions, are recurrent phenomena during evolution, and both of them are involved in reproductive isolation and speciation. To better understand the molecular basis of chromosome rearrangements and their part in karyotype evolution, we have investigated the history of human chromosome 17 by comparative fluorescence in situ hybridization (FISH) and sequence analysis. Results Human bacterial artificial chromosome/p1 artificial ...

  15. Synthesis of novel boron chelate complexes and proposed mechanism of new rearrangement.

    Science.gov (United States)

    Zhang, Rui-Zhe; Feng, Xiao; Liu, Ying; Wang, Sheng-Qing; Liu, Jin-Ting; Zhao, Bao-Xiang

    2015-03-15

    We synthesized novel boron chelate complexes by the reaction of pyrazoline derivatives and boron trifluoride diethyl etherate followed by a new rearrangement. The structures of the compounds were characterized by IR, NMR and HRMS, especially, a typical compound 3c was confirmed by X-ray single crystal analysis. We proposed a mechanism of the rearrangement. Moreover, the absorption and fluorescence spectroscopy of these compounds were measured.

  16. Wagner–Meerwein-Type Rearrangements of Germapolysilanes - A Stable Ion Study

    Science.gov (United States)

    2015-01-01

    The rearrangement of tris(trimethylsilyl)silyltrimethylgermane 1 to give tetrakis(trimethylsilyl)germane 2 was investigated as a typical example for Lewis acid catalyzed Wagner–Meerwein-type rearrangements of polysilanes and polygermasilanes. Direct 29Si NMR spectroscopic evidence is provided for several cationic intermediates during the reaction. The identity of these species was verified by independent synthesis and NMR characterization, and their transformation was followed by NMR spectroscopy. PMID:26294805

  17. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody...

  18. Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

    Science.gov (United States)

    Egan, Jan B; Barrett, Michael T; Champion, Mia D; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K Martin; Boczek, Nicole J; Fonseca, Rafael; Craig, David W; Carpten, John D; Borad, Mitesh J; Stewart, A Keith

    2014-01-01

    Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

  19. Whole genome analyses of a well-differentiated liposarcoma reveals novel SYT1 and DDR2 rearrangements.

    Directory of Open Access Journals (Sweden)

    Jan B Egan

    Full Text Available Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.

  20. Progressive Rearrangement of Telomeric Sequences Added to Both the ITR Ends of the Yeast Linear pGKL Plasmid

    Directory of Open Access Journals (Sweden)

    Gunge Norio

    2003-01-01

    Full Text Available Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. In Saccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid which carried the host telomeric repeats TG1-3 of 300-350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat, suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG1-3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids.

  1. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  2. Chromosomal rearrangements directly cause underdominant F1 pollen sterility in Mimulus lewisii-Mimulus cardinalis hybrids.

    Science.gov (United States)

    Stathos, Angela; Fishman, Lila

    2014-11-01

    Chromosomal rearrangements can contribute to the evolution of postzygotic reproductive isolation directly, by disrupting meiosis in F1 hybrids, or indirectly, by suppressing recombination among genic incompatibilities. Because direct effects of rearrangements on fertility imply fitness costs during their spread, understanding the mechanism of F1 hybrid sterility is integral to reconstructing the role(s) of rearrangements in speciation. In hybrids between monkeyflowers Mimulus cardinalis and Mimulus lewisii, rearrangements contain all quantitative trait loci (QTLs) for both premating barriers and pollen sterility, suggesting that they may have facilitated speciation in this model system. We used artificial chromosome doubling and comparative mapping to test whether heterozygous rearrangements directly cause underdominant male sterility in M. lewisii-M. cardinalis hybrids. Consistent with a direct chromosomal basis for hybrid sterility, synthetic tetraploid F1 s showed highly restored fertility (83.4% pollen fertility) relative to diploids F1 s (36.0%). Additional mapping with Mimulus parishii-M. cardinalis and M. parishii-M. lewisii hybrids demonstrated that underdominant male sterility is caused by one M. lewisii specific and one M. cardinalis specific reciprocal translocation, but that inversions had no direct effects on fertility. We discuss the importance of translocations as causes of reproductive isolation, and consider models for how underdominant rearrangements spread and fix despite intrinsic fitness costs.

  3. Rearrangement dynamics in colloidal particle packings identified through local structure and machine-learning

    Science.gov (United States)

    Davidson, Zoey S.; Still, Tim; Gratale, Matthew D.; Ma, Xiaoguang; Schoenholz, Samuel S.; Sussman, Daniel M.; Liu, A. J.; Yodh, A. G.

    We explore the connection between measures of local structure and particle rearrangements in soft thermal quasi-two-dimensional colloidal systems employing a machine learning approach. Local structure is characterized by two and three point structure functions that measure radial and angular distributions of particles, and rearrangements are identified by a measure of change in average colloidal particle position. By generating labeled training data, we can extract the features of these functions that contribute to the likelihood of a rearrangement. In particular, we use a machine-learning algorithm to construct a decision function in the form of a scalar field we call softness that with high accuracy labels regions of particles more likely to rearrange. Thus, we can predict dynamic rearrangements from the instantaneous local structure. The softness field remains a good predictor when we vary the packing fraction between training and test data sets. In glassy samples, the softness field can identify aging as particles become less likely to undergo cage rearrangements. We gratefully acknowledge financial support through NSF DMR12-05463, MRSEC DMR11-20901, NASA NNX08AO0G, and DE-FG02-05ER46199.

  4. Prevalence of chromosomal rearrangements involving non-ETS genes in prostate cancer.

    Science.gov (United States)

    Kluth, Martina; Galal, Rami; Krohn, Antje; Weischenfeldt, Joachim; Tsourlakis, Christina; Paustian, Lisa; Ahrary, Ramin; Ahmed, Malik; Scherzai, Sekander; Meyer, Anne; Sirma, Hüseyin; Korbel, Jan; Sauter, Guido; Schlomm, Thorsten; Simon, Ronald; Minner, Sarah

    2015-04-01

    Prostate cancer is characterized by structural rearrangements, most frequently including translocations between androgen-dependent genes and members of the ETS family of transcription factor like TMPRSS2:ERG. In a recent whole genome sequencing study we identified 140 gene fusions that were unrelated to ETS genes in 11 prostate cancers. The aim of the present study was to estimate the prevalence of non-ETS gene fusions. We randomly selected 27 of these rearrangements and analyzed them by fluorescence in situ hybridization (FISH) in a tissue microarray format containing 500 prostate cancers. Using break-apart FISH probes for one fusion partner each, we found rearrangements of 13 (48%) of the 27 analyzed genes in 300-400 analyzable cancers per gene. Recurrent breakage, often accompanied by partial deletion of the genes, was found for NCKAP5, SH3BGR and TTC3 in 3 (0.8%) tumors each, as well as for ARNTL2 and ENOX1 in 2 (0.5%) cancers each. One rearranged tumor sample was observed for each of VCL, ZNF578, IMMP2L, SLC16A12, PANK1, GPHN, LRP1 and ZHX2. Balanced rearrangements, indicating possible gene fusion, were found for ZNF578, SH3BGR, LPR12 and ZHX2 in individual cancers only. The results of the present study confirm that rearrangements involving non-ETS genes occur in prostate cancer, but demonstrate that they are highly individual and typically non-recurrent.

  5. MLL rearrangements in pediatric acute lymphoblastic and myeloblastic leukemias: MLL specific and lineage specific signatures

    Directory of Open Access Journals (Sweden)

    te Kronnie Geertruy

    2009-06-01

    Full Text Available Abstract Background The presence of MLL rearrangements in acute leukemia results in a complex number of biological modifications that still remain largely unexplained. Armstrong et al. proposed MLL rearrangement positive ALL as a distinct subgroup, separated from acute lymphoblastic (ALL and myeloblastic leukemia (AML, with a specific gene expression profile. Here we show that MLL, from both ALL and AML origin, share a signature identified by a small set of genes suggesting a common genetic disregulation that could be at the basis of mixed lineage leukemia in both phenotypes. Methods Using Affymetrix® HG-U133 Plus 2.0 platform, gene expression data from 140 (training set + 78 (test set ALL and AML patients with (24+13 and without (116+65 MLL rearrangements have been investigated performing class comparison (SAM and class prediction (PAM analyses. Results We identified a MLL translocation-specific (379 probes signature and a phenotype-specific (622 probes signature which have been tested using unsupervised methods. A final subset of 14 genes grants the characterization of acute leukemia patients with and without MLL rearrangements. Conclusion Our study demonstrated that a small subset of genes identifies MLL-specific rearrangements and clearly separates acute leukemia samples according to lineage origin. The subset included well-known genes and newly discovered markers that identified ALL and AML subgroups, with and without MLL rearrangements.

  6. Evolutionary dynamics of a mitochondrial rearrangement "hot spot" in the Hymenoptera.

    Science.gov (United States)

    Dowton, M; Austin, A D

    1999-02-01

    The arrangement of tRNA genes at the junction of the cytochrome oxidase II and ATPase 8 genes was examined across a broad range of Hymenoptera. Seven distinct arrangements of tRNA genes were identified among a group of wasps that have diverged over the last 180 Myr (suborder Apocrita); many of the rearrangements represent evolutionarily independent events. Approximately equal proportions of local rearrangements, inversions, and translocations were observed, in contrast to vertebrate mitochondria, in which local rearrangements predominate. Surprisingly, homoplasy was evident among certain types of rearrangement; a reversal of the plesiomorphic gene order has arisen on three separate occasions in the Insecta, while the tRNA(H) gene has been translocated to this locus on two separate occasions. Phylogenetic analysis indicates that this gene translocation is real and is not an artifactual translocation resulting from the duplication of a resident tRNA gene followed by mutation of the anticodon. The nature of the intergenic sequences surrounding this region does not indicate that it should be especially prone to rearrangement; it does not generally have the tandem or inverted repeats that might facilitate this plasticity. Intriguingly, these findings are consistent with the view that during the evolution of the Hymenoptera, rearrangements increased at the same time that the rate of point mutations and compositional bias also increased. This association may direct investigations into mitochondrial genome plasticity in other invertebrate lineages.

  7. Nuclear dynamics and genetic rearrangement in heterokaryotic colonies of Fusarium oxysporum.

    Science.gov (United States)

    Shahi, Shermineh; Beerens, Bas; Bosch, Martin; Linmans, Jasper; Rep, Martijn

    2016-06-01

    Recent studies have shown horizontal transfer of chromosomes to be a potential key contributor to genome plasticity in asexual fungal pathogens. However, the mechanisms behind horizontal chromosome transfer in eukaryotes are not well understood. Here we investigated the role of conidial anastomosis in heterokaryon formation between incompatible strains of Fusarium oxysporum and determined the importance of heterokaryons for horizontal chromosome transfer. Using live-cell imaging we demonstrate that conidial pairing of incompatible strains under carbon starvation can result in the formation of viable heterokaryotic hyphae in F. oxysporum. Nuclei of the parental lines presumably fuse at some stage as conidia with a single nucleus harboring both marker histones (GFP- and RFP-tagged) are produced. Upon colony formation, this hybrid offspring is subject to progressive and gradual genome rearrangement. The parental genomes appear to become spatially separated and RFP-tagged histones, deriving from one of the strains, Fol4287, are eventually lost. With a PCR-based method we showed that markers for most of the chromosomes of this strain are lost, indicating a lack of Fol4287 chromosomes. This leaves offspring with the genomic background of the other strain (Fo47), but in some cases together with one or two chromosomes from Fol4287, including the chromosome that confers pathogenicity towards tomato.

  8. ATR-16 due to a de novo complex rearrangement of chromosome 16.

    Science.gov (United States)

    Gallego, Marta S; Zelaya, Gabriela; Feliu, Aurora S; Rossetti, Liliana; Shaffer, Lisa G; Bailey, Kristen A; Bacino, Carlos A; Barreiro, Cristina Z

    2005-01-01

    We describe a child with ATR-16 [alpha-thalassemia (thal)/mental retardation], who was referred for genetic evaluation because of minor anomalies and developmental delay. Cytogenetic analysis demonstrated a de novo complex rearrangement of chromosome 16. Fluorescence in situ hybridization (FISH) analysis, using chromosome 16 subtelomeric probes, showed that this patient had a deletion of the distal short arm of chromosome 16 that contains the alpha-globin genes and a duplication of 16q. Analysis of the alpha-globin locus by Southern blot showed a half normal dose of the alpha-globin gene. Microsatellite marker studies revealed that the duplicated 16q region was maternal in origin. Hematological studies revealed anemia, hypochromia and occasional cells with Hb H inclusion bodies. A hematological screening for alpha-thal should be considered in patients with mild developmental delay and a suggestive phenotype of ATR-16 with microcytic hypochromic anemia and normal iron status. The stellate pattern of the iris, a new finding in our patient, may contribute to a better clinical delineation of both syndromes, ATR-16 and/or duplication of 16qter.

  9. Differential Metabolic Rearrangements after Cold Storage Are Correlated with Chilling Injury Resistance of Peach Fruits

    Science.gov (United States)

    Bustamante, Claudia A.; Monti, Laura L.; Gabilondo, Julieta; Scossa, Federico; Valentini, Gabriel; Budde, Claudio O.; Lara, María V.; Fernie, Alisdair R.; Drincovich, María F.

    2016-01-01

    Reconfiguration of the metabolome is a key component involved in the acclimation to cold in plants; however, few studies have been devoted to the analysis of the overall metabolite changes after cold storage of fruits prior to consumption. Here, metabolite profiling of six peach varieties with differential susceptibility to develop mealiness, a chilling-injury (CI) symptom, was performed. According to metabolic content at harvest; after cold treatment; and after ripening, either following cold treatment or not; peach fruits clustered in distinct groups, depending on harvest-time, cold treatment, and ripening state. Both common and distinct metabolic responses among the six varieties were found; common changes including dramatic galactinol and raffinose rise; GABA, Asp, and Phe increase; and 2-oxo-glutarate and succinate decrease. Raffinose content after long cold treatment quantitatively correlated to the degree of mealiness resistance of the different peach varieties; and thus, raffinose emerges as a candidate biomarker of this CI disorder. Xylose increase after cold treatment was found only in the susceptible genotypes, indicating a particular cell wall reconfiguration of these varieties while being cold-stored. Overall, results indicate that peach fruit differential metabolic rearrangements due to cold treatment, rather than differential metabolic priming before cold, are better related with CI resistance. The plasticity of peach fruit metabolism renders it possible to induce a diverse metabolite array after cold, which is successful, in some genotypes, to avoid CI. PMID:27746802

  10. Mitochondrial fission factor Drp1 maintains oocyte quality via dynamic rearrangement of multiple organelles.

    Science.gov (United States)

    Udagawa, Osamu; Ishihara, Takaya; Maeda, Maki; Matsunaga, Yui; Tsukamoto, Satoshi; Kawano, Natsuko; Miyado, Kenji; Shitara, Hiroshi; Yokota, Sadaki; Nomura, Masatoshi; Mihara, Katsuyoshi; Mizushima, Noboru; Ishihara, Naotada

    2014-10-20

    Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.

  11. Variation induced by DNA rearrangement in a transgenic Bt+CpTI cotton strain

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In the development of transgenic Bt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T4 strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2-1/3 as large as that of the wild transgenic Bt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n = 52; however, there are 4 - 8 univalents observed in meiosis Ⅰ of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCR amplification revealed that Bt and CpTI genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant phenotype might result from either the DNA deletion or T-DNA trans-ferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homo-zygous transgenic line occurred. Further analysis is ongoing.

  12. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model.

    Science.gov (United States)

    Andey, Terrick; Marepally, Srujan; Patel, Apurva; Jackson, Tanise; Sarkar, Shubhashish; O'Connell, Malaney; Reddy, Rakesh C; Chellappan, Srikumar; Singh, Pomila; Singh, Mandip

    2014-06-28

    The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h resulting in a 6

  13. The molecular heterogenity of MLL-rearranged pediatric AML

    NARCIS (Netherlands)

    E.A. Coenen (Eva)

    2013-01-01

    textabstractContinuous renewal of blood cells, so called hematopoiesis, is essential for human life. Starting from several months after birth the majority of blood cell production occurs in the bone marrow. Circulating blood cells are of diverse morphology and function and can be divided in red bloo

  14. Unraveling Mill-Rearranged Pedriatic Acute Myeloid Leukemia

    NARCIS (Netherlands)

    B.V. Balgobind (Brian)

    2011-01-01

    textabstractBlood cells are derived from hematopoietic stem cells (HSC) that reside in the bone marrow. HSC’s are multipotent and have the capacity to diff erentiate into the cells of all blood lineages, i.e. erythrocytes, platelets, neutrophils, eosinophils, basophils, monocytes, T and B lymphocyte

  15. Mycoreovirus genome alterations: similarities to and differences from rearrangements reported for other reoviruses

    Directory of Open Access Journals (Sweden)

    Toru eTanaka

    2012-06-01

    Full Text Available The family Reoviridae is one of the largest virus families with genomes composed of 9 to 12 double-stranded RNA segments. It includes members infecting organisms from protists to humans. It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. Recently distinct genetic alterations were reported for members of the genus Mycoreovirus, Mycoreovirus 1 (MyRV1 and MyRV3 with 11 (S1–S11 and 12 genome segments (S1–S12, respectively. While MyRV3 S8 is lost during subculturing of infected host fungal strains, MyRV1 rearrangements undergo alterations spontaneously and inducibly. The inducible MyRV1 rerarrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus Cryphonectria parasitica hypovirus 1 (CHV1. A total of 5 MyRV1 variants with genome rearranged segments (S1-S3, S6 and S10 are generated in the background of a single viral strain in the presence of CHV1 p29 supplied either transgenically or by coinfection. MyRV1 S4 and S10 are rearranged, albeit very infrequently, in a CHV1-p29 independent fashion. A variant of MyRV1 with substantial deletions in both S4 and S10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type MyRV1. In vivo and in vitro interactions of CHV1 p29 and MyRV1 VP9 are implicated in the induction of MyRV1 rearrangements. However, the mechanism underlying p29-mediated rearrangements remains largely unknown. MyRV1 S4 rearrangements spontaneously occurred independently of CHV1 p29. In the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these MyRV1 and MyRV3 variants contribute to functional analyses of the protein products encoded by those rearranged

  16. Rates of gene rearrangement and nucleotide substitution are correlated in the mitochondrial genomes of insects.

    Science.gov (United States)

    Shao, Renfu; Dowton, Mark; Murrell, Anna; Barker, Stephen C

    2003-10-01

    A number of studies indicated that lineages of animals with high rates of mitochondrial (mt) gene rearrangement might have high rates of mt nucleotide substitution. We chose the hemipteroid assemblage and the Insecta to test the idea that rates of mt gene rearrangement and mt nucleotide substitution are correlated. For this purpose, we sequenced the mt genome of a lepidopsocid from the Psocoptera, the only order of hemipteroid insects for which an entire mtDNA sequence is not available. The mt genome of this lepidopsocid is circular, 16,924 bp long, and contains 37 genes and a putative control region; seven tRNA genes and a protein-coding gene in this genome have changed positions relative to the ancestral arrangement of mt genes of insects. We then compared the relative rates of nucleotide substitution among species from each of the four orders of hemipteroid insects and among the 20 insects whose mt genomes have been sequenced entirely. All comparisons among the hemipteroid insects showed that species with higher rates of gene rearrangement also had significantly higher rates of nucleotide substitution statistically than did species with lower rates of gene rearrangement. In comparisons among the 20 insects, where the mt genomes of the two species differed by more than five breakpoints, the more rearranged species always had a significantly higher rate of nucleotide substitution than the less rearranged species. However, in comparisons where the mt genomes of two species differed by five or less breakpoints, the more rearranged species did not always have a significantly higher rate of nucleotide substitution than the less rearranged species. We tested the statistical significance of the correlation between the rates of mt gene rearrangement and mt nucleotide substitution with nine pairs of insects that were phylogenetically independent from one another. We found that the correlation was positive and statistically significant (R2 = 0.73, P = 0.01; Rs = 0.67, P

  17. Electric Signals Regulate the Directional Migration of Oligodendrocyte Progenitor Cells (OPCs) via β1 Integrin.