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1
Mercatelli, Daniele, Nicola Balboni, Alessandro Palma, Emanuela Aleo, Pietro Paolo Sanna, Giovanni Perini, and Federico Manuel Giorgi. "Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines." Biomolecules 11, no. 2 (January 28, 2021): 177. http://dx.doi.org/10.3390/biom11020177.
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Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.
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Gordon, Mary Contini. "He’s Got Rhythm: the Life and Career of Gene Kelly." Oral History Review 47, no. 1 (November 5, 2019): 133–34. http://dx.doi.org/10.1080/00940798.2019.1673644.
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Stolze, Ineke, Utta Berchner-Pfannschmidt, Patricia Freitag, Christoph Wotzlaw, Jochen Rössler, Stilla Frede, Helmut Acker, and Joachim Fandrey. "Hypoxia-inducible erythropoietin gene expression in human neuroblastoma cells." Blood 100, no. 7 (October 1, 2002): 2623–28. http://dx.doi.org/10.1182/blood-2001-12-0169.
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Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O2)-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O2 values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O2-sensitive α subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the β subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1α, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4α (HNF4α) was found to be indispensable for hypoxia-induced EPO gene expression in hepatoma cells. Western blot analysis and polymerase chain reaction assessment showed that NB cells express neither HNF4α nor the splicing variant HNF4α7 and thus express EPO in an HNF4α-independent manner. Together, SH-SY5Y and Kelly cells may provide a new in vitro model for studying the mechanism of tissue-specific, hypoxia-inducible EPO gene expression.
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Lindberg, Julianne. "The Time of Your Life: Gene Kelly, working-class masculinity, and music." Studies in Musical Theatre 10, no. 2 (June 1, 2016): 177–93. http://dx.doi.org/10.1386/smt.10.2.177_1.
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Yang, Xiaofeng, Taissia G. Popova, Martin S. Goldberg, and Michael V. Norgard. "Influence of Cultivation Media on Genetic Regulatory Patterns in Borrelia burgdorferi." Infection and Immunity 69, no. 6 (June 1, 2001): 4159–63. http://dx.doi.org/10.1128/iai.69.6.4159-4163.2001.
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ABSTRACT Barbour-Stoenner-Kelly II (BSKII) medium and BSKH medium both are routinely used for the cultivation of Borrelia burgdorferi. However, heretofore there have been no studies to compare how these two media affect gene expression patterns in virulentB. burgdorferi. In the present study, we found that someB. burgdorferi strain 297 genes (e.g.,ospA, mlp-7A, mlp-8,p22, and lp6.6) that typically are regulated by temperature or pH displayed their predicted pattern of expression when B. burgdorferi was cultivated in BSKH medium; this was not true when spirochetes were cultivated in conventional BSKII medium. The results suggest that BSKH medium is superior to BSKII medium for gene expression studies with B. burgdorferi.
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Pattullo, Lauren. "Narrative and spectacle in the Hollywood musical: contrasting the choreography of Busby Berkeley and Gene Kelly." Research in Dance Education 8, no. 1 (April 2007): 73–85. http://dx.doi.org/10.1080/14647890701272878.
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Kraut, Anthea. "The Hollywood Dance-In: Abstract and Material Relations of Corporeal Reproduction." Arts 8, no. 4 (October 14, 2019): 133. http://dx.doi.org/10.3390/arts8040133.
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This essay asks what the figure of the Hollywood dance-in—a dancer who performed in place of a star prior to filming and who assisted the choreographer in the creation of dance numbers—can reveal about the reproduction of corporeality as an operation that is both abstract and material. Focusing on the white film star Gene Kelly and his Mexican-born dance-in Alex Romero, the essay shows how the men functioned as literal and virtual doubles for one another in the rehearsal process and argues for an understanding of their relations of reproduction as queer and racially charged.
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Blount, Stacye A. "Book Review: The Material Gene: Gender, Race, and Heredity after the Human Genome Project by Kelly Happe." Gender & Society 28, no. 4 (February 21, 2014): 637–38. http://dx.doi.org/10.1177/0891243214524624.
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Dedoni, Simona, Luisa Marras, Maria C. Olianas, Angela Ingianni, and Pierluigi Onali. "The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7790. http://dx.doi.org/10.3390/ijms22157790.
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Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.
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Haigh, Andrew J., and Vett K. Lloyd. "Loss of genomic imprinting in Drosophila clones." Genome 49, no. 8 (August 1, 2006): 1043–46. http://dx.doi.org/10.1139/g06-042.
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Genomic imprinting is a process that genetically distinguishes maternal and paternal genomes, and can result in parent-of-origin-dependent monoallelic expression of a gene that is dependent on the parent of origin. As such, an otherwise functional maternally inherited allele may be silenced so that the gene is expressed exclusively from the paternal allele, or vice versa. Once thought to be restricted to mammals, genomic imprinting has been documented in angiosperm plants (J.L. Kermicle. 1970. Genetics, 66: 6985), zebrafish (C.C. Martin and R. McGowan. 1995. Genet. Res. 65: 2128), insects, and C. elegans (C.J. Bean, C.E. Schaner, and W.G. Kelly. 2004. Nat. Genet. 36: 100105.). In each case, it appears to rely on differential chromatin structure. Aberrant imprinting has been implicated in various human cancers and has been detected in a number of cloned mammals, potentially limiting the usefulness of somatic nuclear transfer. Here we show that genomic imprinting associated with a mini-X chromosome is lost in Drosophila melanogaster clones.Key words: cloning, Drosophila, genomic imprinting, nuclear transfer.
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Toulza, Pierre-Olivier. "Un Américain « plus parisien que la plupart des Parisiens « : la popularité de Gene Kelly en France dans les années 1950." Contemporary French and Francophone Studies 19, no. 1 (December 20, 2014): 106–20. http://dx.doi.org/10.1080/17409292.2015.982436.
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Genné, Beth. "“FREEDOM INCARNATE”: JEROME ROBBINS, GENE KELLY, AND THE DANCING SAILOR AS AN ICON OF AMERICAN VALUES IN WORLD WAR II." Dance Chronicle 24, no. 1 (April 30, 2001): 83–103. http://dx.doi.org/10.1081/dnc-100103142.
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Jedrusik, M. A., and E. Schulze. "A single histone H1 isoform (H1.1) is essential for chromatin silencing and germline development in Caenorhabditis elegans." Development 128, no. 7 (April 1, 2001): 1069–80. http://dx.doi.org/10.1242/dev.128.7.1069.
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In remarkable contrast to somatic cells, the germline of the nematode Caenorhabditis elegans efficiently silences transgenic DNA. The molecular mechanisms responsible for this have been shown to implicate chromatin proteins encoded by the mes genes (Kelly, W. G. and Fire, A. (1998) Development 125, 2451–2456), of which two are the C. elegans homologs of Polycomb Group gene transcriptional repressors. We have analyzed the contribution of the histone H1 gene family to this specific aspect of germ cells in C. elegans. We show with isotype-specific double stranded RNA-mediated interference (RNAi) that a single member of this gene family (H1.1) is essential for the repression of a silenced reporter-transgene in the germline of hermaphrodites and males, whereas no change is found in the somatic expression of this reporter. Additionally, RNA-mediated interference with H1.1 gene expression can cause a phenotype with severe affection of germline proliferation and differentiation in the hermaphrodite, and even sterility (5%-11% penetrance). These and further features observed in histone H1.1 RNAi experiments are also characteristic of the mes phenotype (Garvin, C., Holdeman, R. and Strome, S. (1998) Genetics 148, 167–185), which is believed to result from the desilencing of genes required for somatic differentiation in the germline. Our observations therefore support this interpretation of the mes phenotype and they identify a single histone H1 isoform (H1.1) as a new component specifically involved in chromatin silencing in the germline of C. elegans.
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Becker, Svea, and Bruce Williams. "What ever happened toWest Side Story? Gene Kelly, jazz dance, and not so real men in Jacques Demy'sThe Young Girls of Rochefort." New Review of Film and Television Studies 6, no. 3 (December 2008): 303–21. http://dx.doi.org/10.1080/17400300802418610.
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Lee, Pui Y., Yutaro Kumagai, Yi Li, Osamu Takeuchi, Hideo Yoshida, Jason Weinstein, Erinn S. Kellner, et al. "TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus." Journal of Experimental Medicine 205, no. 13 (December 1, 2008): 2995–3006. http://dx.doi.org/10.1084/jem.20080462.
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Increased type I interferon (IFN-I) production and IFN-stimulated gene (ISG) expression are linked to the pathogenesis of systemic lupus erythematosus (SLE). Although the mechanisms responsible for dysregulated IFN-I production in SLE remain unclear, autoantibody-mediated uptake of endogenous nucleic acids is thought to play a role. 2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane) induces a lupus-like disease in mice characterized by immune complex nephritis with autoantibodies to DNA and ribonucleoproteins. We recently reported that TMPD also causes increased ISG expression and that the development of the lupus is completely dependent on IFN-I signaling (Nacionales, D.C., K.M. Kelly-Scumpia, P.Y. Lee, J.S. Weinstein, R. Lyons, E. Sobel, M. Satoh, and W.H. Reeves. 2007. Arthritis Rheum. 56:3770–3783). We show that TMPD elicits IFN-I production, monocyte recruitment, and autoantibody production exclusively through a Toll-like receptor (TLR) 7– and myeloid differentiation factor 88 (MyD88)–dependent pathway. In vitro studies revealed that TMPD augments the effect of TLR7 ligands but does not directly activate TLR7 itself. The effects of TMPD were amplified by the Y-linked autoimmune acceleration cluster, which carries a duplication of the TLR7 gene. In contrast, deficiency of Fcγ receptors (FcγRs) did not affect the production of IFN-I. Collectively, the data demonstrate that TMPD-stimulated IFN-I production requires TLR7/MyD88 signaling and is independent of autoantibody-mediated uptake of ribonucleoproteins by FcγRs.
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Fesler, Melissa C., Marianne J. Middelveen, Jennie M. Burke, and Raphael B. Stricker. "Erosive Vulvovaginitis Associated With Borrelia burgdorferi Infection." Journal of Investigative Medicine High Impact Case Reports 7 (January 2019): 232470961984290. http://dx.doi.org/10.1177/2324709619842901.
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We describe a case of acute erosive vulvovaginitis accompanying Borrelia burgdorferi infection. The patient is a 57-year-old woman previously diagnosed with Lyme disease who presented with a painful erosive genital lesion. At the time of the outbreak, she was being treated with oral antibiotics, and she tested serologically positive for B burgdorferi and serologically negative for syphilis. Histological examination of biopsy tissue from the lesion was not characteristic of dermatopathological patterns typical of erosive vulvar conditions. Dieterle-stained biopsy sections revealed visible spirochetes throughout the stratum spinosum and stratum basale, and anti– B burgdorferi immunostaining was positive. Motile spirochetes were observed by darkfield microscopy and cultured in Barbour-Stoner-Kelly–complete medium inoculated with skin scrapings from the lesion. Cultured spirochetes were identified genetically as B burgdorferi sensu stricto by polymerase chain reaction, while polymerase chain reaction amplification of treponemal gene targets was negative. The condition resolved after treatment with additional systemic antibiotic therapy and topical antibiotics. In cases of genital ulceration that have no identifiable etiology, the possibility of B burgdorferi spirochetal infection should be considered.
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Kraut, Anthea. "Female Surrogate Labor and White Corporeal Debt in Singin’ in the Rain." Camera Obscura: Feminism, Culture, and Media Studies 36, no. 2 (September 1, 2021): 1–31. http://dx.doi.org/10.1215/02705346-9052774.
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Abstract This essay revisits the question of credit and debt in the celebrated 1952 film musical Singin’ in the Rain (dir. Gene Kelly and Stanley Donen, US) to show how white women's dancing bodies participate in the “talent relocations” that the movie both thematizes and suppresses. Specifically, it focuses on the relationship between Debbie Reynolds, who was a novice dancer when she was cast in the film, and the two other white women dancers who helped shape Reynolds's filmic body: assistant choreographer Carol Haney and dance-in Jeanne Coyne. Combining feminist and critical race perspectives with production studies, film studies, and dance and performance studies, the essay unites often disconnected gendered and racial analyses of the film by emphasizing the gendered forms of labor and the multiracial genealogies through which dance is reproduced. It also shows how the guise of white credibility enabled Reynolds to conceal her intercorporeal and multiracial debts. Finally, the essay argues that the presence of dancers of color in the film, most notably Rita Moreno, haunts the chains of white corporeal debt that bind Reynolds to Haney and Coyne.
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Lenardo, M. J., K. M. Esser, A. M. Moon, L. H. Van der Ploeg, and J. E. Donelson. "Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated." Molecular and Cellular Biology 6, no. 6 (June 1986): 1991–97. http://dx.doi.org/10.1128/mcb.6.6.1991.
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During the metacyclic stage in the life cycle of Trypanosoma brucei subsp. rhodesiense, the expression of variant surface glycoproteins (VSGs) is restricted to a small subset of antigenic types. Previously we identified cDNAs for the VSGs expressed in metacyclic variant antigen types (MVATs) 4 and 7 and found that these VSG genes do not rearrange when expressed at the metacyclic stage (M. J. Lenardo, A. C. Rice-Ficht, G. Kelly, K. Esser, and J. E. Donelson, Proc. Nathl. Acad Sci. USA 81:6642-6646, 1984). We now provide further evidence that these genes do not rearrange and demonstrate that their 5' upstream regions lack the 72 to 76-base-pair repeats which are considered the substrate for duplication and transposition events. Pulsed field gradient electrophoresis showed that the MVAT VSG genes were located on the largest chromosome-sized DNA molecules, and the lack of the MVAT 4 gene in one of two different serodemes suggested that one mechanism for the evolution of MVAT repertoires is gene deletion. When MVATs were inoculated into the bloodstream of a mammalian host by a bite from the insect vector, they rapidly switched into nonmetacyclic VSG types. We found that this switch was accomplished by a loss of MVAT RNA concomitant with the loss of metacyclic VSGs. Transcription studies with isolated metacyclic nuclei showed that the MVAT genes were expressed in situ from a single locus and were regulated at the level of transcription.
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Lenardo, M. J., K. M. Esser, A. M. Moon, L. H. Van der Ploeg, and J. E. Donelson. "Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated." Molecular and Cellular Biology 6, no. 6 (June 1986): 1991–97. http://dx.doi.org/10.1128/mcb.6.6.1991-1997.1986.
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During the metacyclic stage in the life cycle of Trypanosoma brucei subsp. rhodesiense, the expression of variant surface glycoproteins (VSGs) is restricted to a small subset of antigenic types. Previously we identified cDNAs for the VSGs expressed in metacyclic variant antigen types (MVATs) 4 and 7 and found that these VSG genes do not rearrange when expressed at the metacyclic stage (M. J. Lenardo, A. C. Rice-Ficht, G. Kelly, K. Esser, and J. E. Donelson, Proc. Nathl. Acad Sci. USA 81:6642-6646, 1984). We now provide further evidence that these genes do not rearrange and demonstrate that their 5' upstream regions lack the 72 to 76-base-pair repeats which are considered the substrate for duplication and transposition events. Pulsed field gradient electrophoresis showed that the MVAT VSG genes were located on the largest chromosome-sized DNA molecules, and the lack of the MVAT 4 gene in one of two different serodemes suggested that one mechanism for the evolution of MVAT repertoires is gene deletion. When MVATs were inoculated into the bloodstream of a mammalian host by a bite from the insect vector, they rapidly switched into nonmetacyclic VSG types. We found that this switch was accomplished by a loss of MVAT RNA concomitant with the loss of metacyclic VSGs. Transcription studies with isolated metacyclic nuclei showed that the MVAT genes were expressed in situ from a single locus and were regulated at the level of transcription.
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Irving, S. G., C. H. June, P. F. Zipfel, U. Siebenlist, and K. Kelly. "Mitogen-induced genes are subject to multiple pathways of regulation in the initial stages of T-cell activation." Molecular and Cellular Biology 9, no. 3 (March 1989): 1034–40. http://dx.doi.org/10.1128/mcb.9.3.1034.
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The delivery of a mitogenic signal to T cells via any one of several cell surface molecules elicits a variety of intracellular responses, some or all of which regulate subsequent gene expression events. The expression of nine novel mitogen-induced genes in response to various T-cell-activating agents was examined to evaluate the diversity of pathways which regulate such genes. The relative contribution of distinct secondary signals, individually or together, to mitogen-stimulated gene induction and the capability of individual genes to respond to the sometimes divergent signals generated from different cell surface structures is addressed. The activation of T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with phytohemagglutinin, induced all nine genes. Thus, stimulation by fully mitogenic agents regardless of cell surface-binding specificity correlated with the expression of all of the genes studied. However, heterogeneous patterns of gene expression, encompassing five regulatory classes, were revealed by the use of phorbol 12-myristate 13-acetate, calcium ionophore, and anti-CD28 monoclonal antibody, agents which mediated only a subset of intracellular events and thus an incomplete mitogenic signal. Interleukin-2 and two novel lymphokines represented one regulatory class that appeared to require unique transcriptional activation signals relative to the other mitogen-induced genes. As demonstrated in the accompanying paper (P. F. Zipfel, S. G. Irving, K. Kelly, and U. Siebenlist, Mol. Cell. Biol. 9:1041-1048, 1989), the immediate transcriptional response of T cells to mitogenic stimulation is quite complex, involving numerous genes beyond those which have been previously described. Furthermore, the discrimination of several regulatory phenotypes among these nine genes suggests that a multiplicity of signaling pathways extends from the cell surface to the level of transcription.
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Irving, S. G., C. H. June, P. F. Zipfel, U. Siebenlist, and K. Kelly. "Mitogen-induced genes are subject to multiple pathways of regulation in the initial stages of T-cell activation." Molecular and Cellular Biology 9, no. 3 (March 1989): 1034–40. http://dx.doi.org/10.1128/mcb.9.3.1034-1040.1989.
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The delivery of a mitogenic signal to T cells via any one of several cell surface molecules elicits a variety of intracellular responses, some or all of which regulate subsequent gene expression events. The expression of nine novel mitogen-induced genes in response to various T-cell-activating agents was examined to evaluate the diversity of pathways which regulate such genes. The relative contribution of distinct secondary signals, individually or together, to mitogen-stimulated gene induction and the capability of individual genes to respond to the sometimes divergent signals generated from different cell surface structures is addressed. The activation of T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with phytohemagglutinin, induced all nine genes. Thus, stimulation by fully mitogenic agents regardless of cell surface-binding specificity correlated with the expression of all of the genes studied. However, heterogeneous patterns of gene expression, encompassing five regulatory classes, were revealed by the use of phorbol 12-myristate 13-acetate, calcium ionophore, and anti-CD28 monoclonal antibody, agents which mediated only a subset of intracellular events and thus an incomplete mitogenic signal. Interleukin-2 and two novel lymphokines represented one regulatory class that appeared to require unique transcriptional activation signals relative to the other mitogen-induced genes. As demonstrated in the accompanying paper (P. F. Zipfel, S. G. Irving, K. Kelly, and U. Siebenlist, Mol. Cell. Biol. 9:1041-1048, 1989), the immediate transcriptional response of T cells to mitogenic stimulation is quite complex, involving numerous genes beyond those which have been previously described. Furthermore, the discrimination of several regulatory phenotypes among these nine genes suggests that a multiplicity of signaling pathways extends from the cell surface to the level of transcription.
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LEE, Gha Young, Nam Hee KIM, Zheng-Shan ZHAO, Bong Soo CHA, and Y. Sam KIM. "Peroxisomal-proliferator-activated receptor alpha activates transcription of the rat hepatic malonyl-CoA decarboxylase gene: a key regulation of malonyl-CoA level." Biochemical Journal 378, no. 3 (March 15, 2004): 983–90. http://dx.doi.org/10.1042/bj20031565.
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MCD (malonyl-CoA decarboxylase), which catalyses decarboxylation of malonyl-CoA, is known to play an important role in the regulation of malonyl-CoA concentration. Recently, it has been observed that the expression of MCD is significantly decreased in the hearts of the PPARα (peroxisome-proliferator-activated receptor α) (−/−) mice, where the rate of fatty-acid oxidation is decreased by the increased malonyl-CoA level [Campbell, Kozak, Wagner, Altarejos, Dyck, Belke, Severson, Kelly and Lopaschuk (2002) J. Biol. Chem. 277, 4098–4103]. This suggests that MCD may be transcriptionally regulated by PPARα. To investigate whether PPARα is truly responsible for transcriptional regulation of the rat MCD gene, transient reporter assay was performed in CV-1 cells. The promoter activity was increased by 17-fold in CV-1 cells co-transfected with PPARα/retinoid X receptor α expression plasmid. In sequence analysis of the promoter region, three putative PPREs (PPAR response elements) were identified, and promoter deletion analysis showed that PPRE2 and PPRE3 were functional. Electrophoretic mobility-shift assays revealed that PPARα/retinoid X receptor α heterodimer indeed bound to the two PPREs, and the binding specificity of PPARα on PPRE was also confirmed by experiments with mutated oligonucleotides. These results indicate that the elements behaved as a responsive site to PPARα activation. MCD mRNA levels in WY14643-treated rat hepatoma cells as well as in the liver of fenofibrate-fed Otsuka Long-Evans Tokushima fatty rats were also found to be increased, suggesting that PPARα can activate the rat hepatic MCD transcription by binding to the PPREs in the promoter. We propose that MCD performs an important role in understanding the regulatory mechanism between activated PPARα and fatty-acid oxidation by altering the malonyl-CoA concentration.
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Muller, Lars U. W., Michael Milsom, Chad E. Harris, Jeff Bailey, and David A. Williams. "The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC." Blood 110, no. 11 (November 16, 2007): 194. http://dx.doi.org/10.1182/blood.v110.11.194.194.
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Abstract Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.
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Cluss, Robert G., Damon A. Silverman, and Thomas R. Stafford. "Extracellular Secretion of the Borrelia burgdorferi Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein." Infection and Immunity 72, no. 11 (November 2004): 6279–86. http://dx.doi.org/10.1128/iai.72.11.6279-6286.2004.
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ABSTRACT Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 107 to 109 cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.
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Güner, Ece S., Naoya Hashimoto, Teruki Kadosaka, Yasuyuki Imai, and Toshiyuki Masuzawa. "A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey." Microbiology 149, no. 9 (September 1, 2003): 2539–44. http://dx.doi.org/10.1099/mic.0.26464-0.
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A novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour–Stoenner–Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44 %) and 72 of 185 nymphs (39 %) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34–37 °C, and it could grow at 39 °C. Doubling times at 34 and 37 °C were 5·3 and 5·1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99·2 to 100 %; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90 %. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.
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Manhes, Caroline, Vincent Goffin, Paul A. Kelly, and Philippe Touraine. "Autocrine prolactin as a promotor of mammary tumour growth." Journal of Dairy Research 72, S1 (July 14, 2005): 58–65. http://dx.doi.org/10.1017/s0022029905001196.
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Prolactin (PRL) plays a key role in normal growth, development and differentiation of the mammary gland. Indeed, strong evidence suggests that the development of alveolar cells requires not only oestradiol and progesterone, but also PRL. In vitro, PRL has mitogenic activity on normal mouse mammary epithelial cells (reviewed in Das & Vonderhaar, 1997). In vivo, PRL also seems to be involved in such proliferative activity, although it is more difficult to distinguish the role of PRL from the influence of the hormonal milieu (Das & Vonderhaar, 1997). This physiological role of PRL in lobular development of the mammary gland is supported by results obtained from mice deficient for PRL (Horseman et al. 1997) or for its receptor (PRLR) (Ormandy et al. 1997). Although the infertility of females homozygous for the deletion of the PRLR gene (PRLR−/−) can be partially reversed by restoring progesterone levels close to normal, their mammary gland fails to differentiate during pregnancy, leading to lactation failure (Binart et al. 2000). In addition, heterozygous mice (PRLR+/−), who have half normal receptor levels, show impaired mammary gland development and fail to lactate following their first pregnancy, clearly indicating that signals mediated by the PRL/PRLR interaction have to achieve a certain level to permit mammary gland differentiation and lactation (Kelly et al. 2002). Since the pioneering work of Topper (Topper, 1970), who observed that PRL was necessary to induce casein synthesis, our understanding of the mechanism of such induction has greatly expanded. PRL appears to be the primary hormone involved in this activity, although other hormones such as insulin and glucocorticoids are also required for lactation.
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Hughes, Nicky J., Chris L. Clayton, Peter A. Chalk, and David J. Kelly. "Helicobacter pylori porCDAB and oorDABCGenes Encode Distinct Pyruvate:Flavodoxin and 2-Oxoglutarate:Acceptor Oxidoreductases Which Mediate Electron Transport to NADP." Journal of Bacteriology 180, no. 5 (March 1, 1998): 1119–28. http://dx.doi.org/10.1128/jb.180.5.1119-1128.1998.
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ABSTRACT Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953–3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por,oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. BothporD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB andoorA appeared to be lethal.
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Crutch, Sebastian J., and Elizabeth K. Warrington. "Taxonomic and Thematic Organisation of Proper Name Conceptual Knowledge." Behavioural Neurology 24, no. 4 (2011): 265–76. http://dx.doi.org/10.1155/2011/563620.
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We report the investigation of the organisation of proper names in two aphasic patients (NBC and FBI). The performance of both patients on spoken word to written word matching tasks was inconsistent, affected by presentation rate and semantic relatedness of the competing responses, all hallmarks of a refractory semantic access dysphasia. In a series of experiments we explored the semantic relatedness effects within their proper name vocabulary, including brand names and person names. First we demonstrated the interaction between very fine grain organisation and personal experience, with one patient with a special interest in the cinema demonstrating higher error rates when identifying the names of actors working in a similar film genre (e.g. action movies: Arnold Schwarzenegger, Bruce Willis, Sylvester Stallone, Mel Gibson) than those working in different genres (e.g. Arnold Schwarzenegger, Gregory Peck, Robin Williams, Gene Kelly). Second we compared directly two potential principles of semantic organisation – taxonomic and thematic. Furthermore we considered these principles of organisation in the context of the individuals' personal knowledge base. We selected topics matching the interests and experience of each patient, namely cinema and literature (NBC) and naval history (FBI). The stimulus items were arranged in taxonomic arrays (e.g. Jane Austen, Emily Bronte, Agatha Christie), thematic arrays (e.g. Jane Austen, Pride and Prejudice, Mr Darcy), and unrelated arrays (e.g. Jane Austen, Wuthering Heights, Hercule Poirot). We documented that different patterns of taxonomic and thematic organisation were constrained by whether the individual has limited knowledge, moderate knowledge or detailed knowledge of a particular vocabulary. It is suggested that moderate proper name knowledge is primarily organised by taxonomy whereas extensive experience results in a more detailed knowledge base in which theme is a powerful organising principle.
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Warnecke, Christina, Zaneta Zaborowska, Jens Kurreck, Volker A. Erdmann, Ulrich Frei, Michael Wiesener, and Kai‐Uwe Eckardt. "Differentiating the functional role of hypoxia‐inducible factor (HIF)‐1α and HIF‐2α (EPAS‐1) by the use of RNA interference: erythropoietin is a HIF‐2α target gene in Hep3B and Kelly cells." FASEB Journal 18, no. 12 (July 2004): 1462–64. http://dx.doi.org/10.1096/fj.04-1640fje.
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Metais, Jean-Yves, Ashley E. Dunfee, Rodrigo T. Calado, and Cynthia E. Dunbar. "BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells." Blood 110, no. 11 (November 16, 2007): 3365. http://dx.doi.org/10.1182/blood.v110.11.3365.3365.
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Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.
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Abar, Bijan, Cambre Kelly, Anh Pham, Nicholas Allen, Helena Barber, Alexander Kelly, Ken Gall, and Samuel Adams. "4464 Effect of Surface Topography on In Vitro and Mechanical Performance of 3D Printed Titanium." Journal of Clinical and Translational Science 4, s1 (June 2020): 130. http://dx.doi.org/10.1017/cts.2020.386.
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OBJECTIVES/GOALS: The goal of the study is to understand how changing the surface roughness of 3D printed Titanium either by processing printed samples or artificially printing rough topography impacts the mechanical and biological properties of the Titanium. METHODS/STUDY POPULATION: Titanium dog bones and discs were printed via laser powder bed fusion. groups were defined as 1. polished, 2.blasted, 4.as built, 4.sprouts and 5.rough sprouts. Roughness was measured with line measurement using a confocal microscope. Tensile testing of dog bones produced stress strain curves. MC3T3 preosteoblast were seeded on discs. Samples were analyzed at 0, 2, and 4 weeks. A cell viability assay and confocal fluorescent microscopy assessed cell growth. Alkaline Phosphatase (ALP) assay and Quantitative Polymerase Chain Reaction (qPCR) examined cell differentiation. Extracellular matrix (ECM) was stained for collagen and calcium. Scanning Electron Microcopy (SEM) was done on sputter coated discs. RESULTS/ANTICIPATED RESULTS: Measured roughness defined by Rz, maximum peak to valley distance of the sample profile ranged from 2.6-65.1 µm. The addition of printed roughness in the sprouts and rough sprouts group significantly diminished ductility resulting in early strain to failure during tensile testing. Cells adhered and proliferated on discs regardless of roughness group. There was no statistical difference in ALP activity, but qPCR showed that rough groups (sprouts and rough sprouts) had diminished Osteocalcin gene expression at week 2 and 4. The ECM in the rough groups was more resistant to repeated washes and was more extensive with SEM. DISCUSSION/SIGNIFICANCE OF IMPACT: Printing roughness diminished mechanical properties without clear benefit to cell growth. Roughness features were on mesoscale, unlike samples in literature on microscale that increase cell activity. Printed topography may aid in implant fixation and not osseous integration as hypothesized. CONFLICT OF INTEREST DESCRIPTION: Dr. Samual Adams, Dr. Ken Gall and Cambre Kelly own stock and/or stock options in restor3d, Inc.
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Lee, S., ED Zambas, WL Marsh, and CM Redman. "The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells." Blood 81, no. 10 (May 15, 1993): 2804–9. http://dx.doi.org/10.1182/blood.v81.10.2804.bloodjournal81102804.
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The Kell blood group is one of the major antigenic systems in human red blood cells. To determine the location of the Kell gene on human chromosomes, panels containing genomic DNA of human-hamster somatic cell hybrids were hybridized with radiolabeled cDNA probe specific for the Kell locus. Only the samples containing DNA from chromosome 7 gave positive hybridization signals. In situ hybridization analysis, using genomic clones isolated with the cDNA, localized the KEL gene to 7q33. Northern blot analysis of poly(A)+ RNA from human brain, kidney, lung, fetal and adult liver, and bone marrow showed that Kell transcripts were only present in fetal liver and bone marrow. This indicates that the Kell protein, which carries the Kell antigens, may only be expressed in erythroid tissues.
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González-Castillo, D. D., R. E. Barbosa-Cobos, G. E. Lugo Zamudio, M. A. Saavedra, R. E. Sánchez-Briones, I. Alemán-Ávila, and J. Ramírez Bello. "AB0009 ASSOCIATION BETWEEN POLYMORPHISMS OF BANK1 AND MANIFESTATIONS OF SLE." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1308.1–1308. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5141.
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Background:BANK1encodes an adapter/scaffold protein primarily expressed in B cells, which is involved in cell signaling and activation. Genome-wide association studies (GWAS) have identified differentBANK1single nucleotide variants (SNVs) associated with SLE primarily in European or Asian-derived populations. Interestingly, we recently have documented an association between this gene and susceptibility to systemic lupus erythematosus (SLE) in Mexican population.Objectives:To determine whether theBANK1R61H (rs10516487G/A) and A383T (rs3733197G/A) SNVs are associated with clinical and immunological manifestations in SLE.Methods:Our study included 123 Mexican women with SLE (SLICC 2012 criteria). Genotyping of the twoBANK1SNVs were obtained by TaqMan probes and real-time PCR. An association study was performed between the alleles and genotypes ofBANK1R61H and A383T with the clinical and immunological manifestations included in the SLE SLICC classification criteria. Hardy-Weinberg equilibrium and an association study was performed using Finetti, apvalue ≤ 0.05 indicated association.Results:We identify an average age of 38.5±12. Cases and controls remained in Hardy-Weinberg equilibrium. An association with susceptibility to SLE was found between genotypes of the twoBANK1SNVs and joint manifestations (rs1051487G/A; AA + GA vs GG, OR 4.45,p=0.004, rs3733197G/A; AA + GA vs GG, OR 2.66,p=0.032, respectively), as well as with protection for neurological and renal involvement (rs1051487G/A, OR 0.16,p=0.02, rs3733197G/A; OR 0.40,p=0.02, respectively) (Table 1a and b). No association was found with other clinical manifestations.Conclusion:Our data in the Mexican population show that bothBANK1R61H and A383T SNVs are risk factors for synovitis. On the other hand, theseBANK1R61H and A383T variants are protective factors for neurological and renal damage, respectively.References:[1]Ramírez-Bello J, Jiménez-Morales S, Montufar-Robles I, et al. BLK and BANK1 polymorphisms and interactions are associated in Mexican patients with systemic lupus erythematosus. Inflamm Res. 2019;68:705-13[2]J. De Azevêdo Silva, C. Addobbati, P. Sandrin-Garcia and S. Crovella. Systemic Lupus Erythematosus: Old and New Susceptibility Genes versus Clinical Manifestations. Current Genomics. 2014;15:52-65[3]Sánchez E, Rasmussen A, Riba L, Acevedo E, Kelly J, Langefeld CD, et al. Impact of Genetic Ancestry and Socio-Demographic Status on the Clinical Expression of Systemic Lupus Erythematosus in Amerindian-European Populations. Arthritis Rheum. 2012; 64(11):3687–3694[4]Castillejo-López C, Delgado-Vega AM, Wojcik J, et al. Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK. Ann Rheum Dis. 2012;71:136–42[5]Kozyrev SV, Abelson AK, Wojcik J, et al. Functional variants in the B-cell gene BANK1 are associated with systemic lupus erythematosus. Nat Genet. 2008;40:211-6Disclosure of Interests:None declared
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Lee, S., ED Zambas, WL Marsh, and CM Redman. "The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells." Blood 81, no. 10 (May 15, 1993): 2804–9. http://dx.doi.org/10.1182/blood.v81.10.2804.2804.
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Abstract The Kell blood group is one of the major antigenic systems in human red blood cells. To determine the location of the Kell gene on human chromosomes, panels containing genomic DNA of human-hamster somatic cell hybrids were hybridized with radiolabeled cDNA probe specific for the Kell locus. Only the samples containing DNA from chromosome 7 gave positive hybridization signals. In situ hybridization analysis, using genomic clones isolated with the cDNA, localized the KEL gene to 7q33. Northern blot analysis of poly(A)+ RNA from human brain, kidney, lung, fetal and adult liver, and bone marrow showed that Kell transcripts were only present in fetal liver and bone marrow. This indicates that the Kell protein, which carries the Kell antigens, may only be expressed in erythroid tissues.
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Daniels, GL, F. Weinauer, C. Stone, M. Ho, CA Green, H. Jahn-Jochem, R. Offner, and AP Monaco. "A combination of the effects of rare genotypes at the XK and KEL blood group loci results in absence of Kell system antigens from the red blood cells." Blood 88, no. 10 (November 15, 1996): 4045–50. http://dx.doi.org/10.1182/blood.v88.10.4045.bloodjournal88104045.
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The 22 antigens of the Kell blood group system are located on a red blood cell (RBC) membrane glycoprotein that shows sequence homology with a family of metalloendopeptidases. Expression of the Kell system antigens is partially governed by XK, an X-linked gene that encodes the Kx protein; absence of Kx results in reduced Kell antigen expression. Almost total absence of Kell antigens from the RBCs of a German man with no symptoms of neuroacanthocytosis could not be due to the Kell- null phenotype, Ko, because his RBCs had very weak expression of Kx antigen and his three children were Kp(a + b+). Kell antigens were normal on the RBCs of his son but weak on those of his two daughters. An Nla III restriction fragment-length polymorphism within the KEL gene showed the Kpa/Kpa genotype in the propositus. Sequencing of his XK gene showed a single base change within the donor splice consensus sequence of intron 2. A BsaAl restriction fragment-length polymorphism showed the mutation in both of his daughters but not in his son. The extreme depression of the Kell antigens of the propositus must be due to a combination of effects, ie, homozygosity for Kpa and deficiency of Kx protein, each of which is capable of causing some degree of weakening of Kell antigens.
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Zvegintseva, Irina A. "A Criminal as the Main Movie Character, or Old Themes and New Solutions." Journal of Flm Arts and Film Studies 8, no. 3 (September 15, 2016): 115–25. http://dx.doi.org/10.17816/vgik83115-125.
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The former British colony, emerged as a place of exile for the most dangerous criminals and unreliable people from the metropolis, Australia began its existence very unenviable, appearing on the world map called "The Earths hell", which was used to frighten children in Europe. The fact is: the gene fund of the nation - the convicts, their guards, and adventurers came from all over Europe in hope of a better life. The first half of the 19th century Australia, in fact, remained a giant reforming home, a jail. And whatever paradoxical it might explain the significant number of films shot in the 20th and in 21st centuries with criminals as protagonists. When touching upon permanent plots and problems in Australian cinema, it should be noted that the "eternal" love of the inhabitants of the Green continent to the favorite national hero Ned Kelly, a former convict and burglar has not disappeared. In the minds of the Australians the burglar has become a symbol of the fighter against injustice, a sort of "Australian Robin Hood". The main characters of the movies were bushrangers in Australia called escaped convicts, pariahs of the society, hunting armed robberies and burglaries, hiding from justice in the vast valleys of the Australian Bush. Here, incidentally, there is a parallel with the American film industry that also has surpasses the rank of the most beloved and popular criminals in the country from Al Capone, Bonnie Parker and Clyde Barrow up to Bugsy Siegel and John Dillinger. But soon such films were banned because of the monopolies of the USA and the UK movies on the Australian market. However, life itself has started to supply filmmakers with the stories that hardly could come to the minds of writers with the wildest imagination. The real horrible crimes and not less real maniacs, sadists, pedophiles, whose actions have forced to shudder the whole society, both in the past and the present, formed the basis of a number of films shot in Australia. The analysis of these movies, the authors' position, the artistic value of works have become the target of this article.
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de Rooij, Jasmijn D. E., Marry M. van den Heuvel-Eibrink, Iris H. I. M. Hollink, Susan T. C. J. M. Arentsen-Peters, H. Berna Beverloo, Janneke F. van Galen, Andre Baruchel, et al. "NUP98/JARID1A Is a New Recurrent Genetic Abnormality in Pediatric Acute Megakaryoblastic Leukemia with a Distinct HOX-Gene Expression Pattern." Blood 120, no. 21 (November 16, 2012): 537. http://dx.doi.org/10.1182/blood.v120.21.537.537.
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Abstract Abstract 537 Introduction: Cure rates in pediatric AML are currently in the 60–70% range despite treatment with intensive chemotherapy. To improve prognosis new treatment targets need to be identified, hence there is a need to better understand the underlying biology. It is hypothesized that AML results from at least two types of mutations which non-randomly collaborate in leukemogenesis. The type-I aberrations confer a proliferative advantage, type-II mutations lead to impairment of hematopoietic differentiation (Kelly et al, 2002). We recently described NUP98/NSD1 as recurrent event in cytogenetically normal AML (Hollink et al, 2011). Patients with NUP98/NSD1 had dismal outcome, and a stem-cell phenotype characterized by overexpression of homeobox (HOX) A and –B genes. Using split-signal FISH on 122 pediatric AML cases without driving oncogenic mutation, 26 NUP98- rearranged cases were identified, including 1 patient with acute megakaryoblastic leukemia (AMKL). We previously reported a patient with fusion of JARID1A, located on chromosome 12p13, to NUP98, located on chromosome 11p15, in a non-Down Syndrome (DS) AMKL case (Van Zutven et al, 2006). Therefore, a large series of non-DS AMKL patients was screened for NUP98/JARID1A and for other abnormalities, including the novel CBFA2T3/GLIS2 translocation (Gruber et al, ASH2011; #757). Methods: Samples from 105 pediatric non-DS AMKL cases, diagnosed between 1998 and 2011, were obtained from the DCOG, the AML-BFM SG, the Saint-Louis Hospital in Paris, and the COG. AMKL is more common in DS patients, therefore we also screened a series of DS AMKL (n=16). Centrally reviewed clinical and cell-biological data were provided by these study groups. Translocation of NUP98/JARID1A, MLL-rearrangements, RBM15/MKL1, and CBFA2T3/GLIS2 were identified using RT-PCR, as well as molecular characterization including hospots for the following mutations: FLT3, KIT, RAS, PTPN11, NPM1, WT1, and CEBPA. HOXA and –B expression levels were analyzed using gene expression profiling (Affymetrix) in 274 pediatric AML patients (Balgobind et al, 2011) including 9 AMKL patients, and validated with quantitative real-time PCR (n=37). Results: NUP98/JARID1A translocations were identified in 11 patients (11%). Four other patients had a NUP98- aberration with unknown translocation partner based on split signal FISH. We identified 16/105 patients with RBM15/MKL1, 13/105 with CBFA2T3/GLIS2 translocation, and 13/96 harbouring an MLL-rearrangement. Hence, specific non-random abnormalities could be defined in 61% of pediatric AMKL cases. Only 3/45 cases harboured a type-I mutation, all localized in the RAS gene. Comparing NUP98/JARID1A positive cases with negative cases in pediatric AMKL, no significant differences in patient characteristics including sex, age, and white blood cell count (WBC) were found. Considering prognosis, 5-year pEFS (22±14% vs. 36±6%, p=0.50) did not differ significantly from all other AMKL patients, nor did the cumulative incidence of relapse (56±19% vs. 54±7% p=0.9). CBFA2T3/GLIS2 translocated patients also did not differ from other AMKL patients (pEFS 19±16% vs. 36±6%, p=0.63). However, 5-year pEFS for RBM15/MKL1 translocated patients was significantly better (73±13% vs. 28±6%, p=0.043), but not in multivariate analysis adjusted for age and WBC. Gene expression analysis showed significantly higher HOXA5/A9/A10 and HOXB2/B3/B4/B5/B6 expression in NUP98/JARID1A compared to other pediatric AML cases. We did not identify any NUP98/JARID1A cases in the 16 DS AMKL patients. Discussion and conclusion: NUP98/JARID1A is a recurrent cryptic translocation in approximately 11% of pediatric AMKL cases. In 61% of all AMKL cases a type-II mutation could now be identified. Similar to NUP98-NSD1 a stem-cell phenotype was detected with persistent HOXAB-gene expression. Although NUP98/JARID1A did not influence prognosis, outcome in pediatric AMKL is unsatisfactory. NUP98 is known to recruit CREBBP/p300 resulting in histone acetylation, and transcriptional activation of HOX genes (Wang et al, 2007), suggesting that histone acetyltransferase inhibitors may be active. Moreover, JARID1A is unable to demethylate H3K4me2/3, which also results in sustained up regulation of HOX genes. This may provide options for targeted therapy. Disclosures: No relevant conflicts of interest to declare.
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Dugo, Matteo, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Robert S. Seitz, Tyler J. Nielsen, et al. "Abstract PD10-06: Predictive value of RT-qPCR 27-gene IO score and comparison with RNA-Seq IO score in the NeoTRIPaPDL1 trial." Cancer Research 82, no. 4_Supplement (February 15, 2022): PD10–06—PD10–06. http://dx.doi.org/10.1158/1538-7445.sabcs21-pd10-06.
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Abstract Background The identification of biomarkers for optimization of immune checkpoint inhibitors (ICI) treatment is an unmet clinical need. In the Phase III randomized trial, NeoTRIPaPDL1, a post-hoc analysis of whole transcriptome RNA-Seq data, previously showed that the 27-gene IO score is a potential predictive biomarker of increased pathological complete response with the addition of atezolizumab to carboplatin/nab-paclitaxel (Bianchini G ESMO 2021). However, the laboratory implementation of gene-expression signatures measured using RNA-seq is challenging. Therefore, we further assessed the predictive value of the IO score using a twenty-seven gene RT-qPCR assay on NeoTRIP samples, and compared to the previously reported RNA-Seq version of the assay. Methods The NeoTRIP study randomized patients to eight cycles of carboplatin/nab-paclitaxel (CT) with or without atezolizumab (CT/A). 258 patients were evaluable for pCR (breast and nodes) as Per-Protocol Population. We assessed the IO score as binary and continuous variables using the CAP/CLIA validated DetermaIO qPCR test (Saltman et al 2021) on pre-treatment core biopsies (n=220/258; 85.3%), all of which have RNA-Seq data available. We evaluated the association between IO score defined by RT-qPCR and RNA-Seq, and the association of the IO score defined by RT-qPCR test with PD-L1 IHC (Ventana SP142), stromal TILs (sTILs), and pCR. Results Comparison of continuous IO scores between the RT-qPCR assay and the RNA-Seq algorithm had a Pearson’s correlation of 0.94 (p < 0.0001). High agreement between categorical IO scores was also observed (Cohens’ kappa = 0.84; 95% confidence interval [CI] = 0.77-0.91; p < 0.0001). RT-qPCR IO score was balanced in the two arms (p = 0.65) with 44% and 40% positive patients in the CT and CT/A arms, respectively. The RT-qPCR IO score was correlated with both PD-L1 (Pearson’s r = 0.64; p < 0.0001) and sTILs (Pearson’s r = 0.67; p < 0.0001). Continuous IO score was significantly predictive of pCR in CT/A (Odds ratio [OR] = 3.12; 95% CI = 1.20-8.10; p<0.019), but not CT arm (OR = 1.28; 95% CI = 0.54-3.01; p = 0.578). Considering the binary IO score, OR were 2.87 [1.27-6.47] (p = 0.011) and 0.91 [0.43-1.93] (p = 0.812), in CT/A and CT, respectively (interaction test p = 0.043). The pCR rate for CT/A vs CT was 69.8% vs 46.9% in IO score positive [+22.9%, p = 0.046, Chi-squared test] and 44.6% vs 49.2% [-4.6%, p = 0.73] in IO score negative. A significant interaction was found between continuous PD-L1 and continuous IO-score (p = 0.006). Among PD-L1-neg, 9 patients were IO score positive (10.1%). The pCR rate in this group was 3/4 (75%) in the CT/A arm and 1/5 (20%) in CT arm. The predictive value of IO score by RT-qPCR was similar to RNA-Seq. Conclusions We observed a high level of agreement and concordance between IO scores assessed by RT-qPCR and RNA-Seq, indicating that the 27-gene IO assay and algorithm is robust and the choice of platform has limited impact. This finding also demonstrates the high quality of NeoTRIP RNA-Seq data. In this post-hoc analysis, IO score assessment by this CLIA validated RT-qPCR test was confirmed to be predictive of atezolizumab benefit over CT alone in a randomized trial. Citation Format: Matteo Dugo, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Robert S. Seitz, Tyler J. Nielsen, Marc Thill, Antonio Anton, Stefania Russo, Eva Maria Ciruelos, Brock L. Schweitzer, Douglas T. Ross, Barbara Galbardi, Richard Greil, Vladimir Semiglazov, Balázs Gyorffy, Marco Colleoni, Catherine Kelly, Gabriella Mariani, Lucia Del Mastro, Pinuccia Valagussa, Giuseppe Viale, Maurizio Callari, Luca Gianni, Giampaolo Bianchini. Predictive value of RT-qPCR 27-gene IO score and comparison with RNA-Seq IO score in the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-06.
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Tambaro, Francesco Paolo, Ilaria Lepore, Carmela Dell Aversana, Floriana De Bellis, Marco Miceli, Vincenzo Carafa, Angela Nebbioso, Felicetto Ferrara, Guillermo garcia Manero, and Lucia Altucci. "BARD1: a New Target In Leukemia." Blood 116, no. 21 (November 19, 2010): 4642. http://dx.doi.org/10.1182/blood.v116.21.4642.4642.
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Abstract Abstract 4642 Background BARD1 (BRCA1-associated RING domain protein 1) was first described as a BRCA1 partner and tumour suppressor protein, muted in most cases of breast and ovarian cancer. BARD1 functions are BRCA1-dependent, requiring formation of a stable heterodimer through interaction of the RING finger domains. BRCA1-independent functions for BARD1 have recently been described, making it an interesting and important molecule for study. BARD1 is expressed in almost all human tissues, including haematological cells, testis and breast tissue, but it is over-expressed in leukaemias, sarcomas and testis cancer, implicating BARD1 in development of cancer. Different BARD1 isoforms are up-regulated in breast, ovarian and uterine cancers but markedly down-regulated or absent in healthy tissues. Therefore, the presence of these isoforms might be a risk factor or causal event in the cancer pathogenesis. We investigated the role of BARD1 isoforms in leukaemia, through the study of the epigenetic mechanisms involved in regulation of its expression and function. As such, we determined the expression of a specific BARD1 isoform in different human leukaemia cell lines the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) of expression of BARD1. Results. Four human leukaemia cell lines (U937, NB4, K562 and HL60) express the BARD1 isoform of interest. Treatment of these cell lines with SAHA at 5 μ M resulted in decreased expression of this isoform (Fig. 1). Decreased expression of BARD1 by SAHA was also observed in in human breast cancer MCF7 cells, the usual model for BARD1 experiments and in the human neuroblastoma cell line Kelly (Fig. 2), but not in HeLa cells or an human epithelial carcinoma cell line (Fig. 2). The reduced expression of BARD1 was attributed to the action of 2 specific micro-RNAs (miRNAs), that directly bind its 3′untranslated region (UTR). SAHA-induced expression of miR-19a and miR-19b in primary human leukemia cells as demonstrated using miRNA microarray expression analysis and confirmed by Real-Time PCR (Fig. 3). These 2 miRNA were up-regulated after SAHA stimulation in human leukaemia cells. BARD1 is the predicted target for miR-19a and miR-19b based on miRBase database analyses. Transient transfection in NB4 cells with mimic miR-19a and mimic miR-19b confirmed expression of these miRNAs was associated with BARD1 down-regulation (Fig. 4 5.). The BARD1 3′UTR was cloned into a pGL3 vector with a downstream the luciferase gene reporting gene (Fig. 6). The plasmid was co-transfected in HeLa cells with each mimic miRNA and a luciferase assay was performed. We demonstrate that expression of miR-19a and miR-19b can directly bind BARD1 3′UTR, reducing luciferase activity in this system (Fig. 7), thereby confirming that BARD1 is a target of these miRNAs. These findings support our hypothesis that BARD1 is a promising target in leukemia and should be further investigated for its diagnostic and prognostic features. Disclosures: No relevant conflicts of interest to declare.
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CAMARA-CLAYETTE, Valérie, Cécile RAHUEL, Claude LOPEZ, Claude HATTAB, Virginie VERKARRE, Olivier BERTRAND, and Jean-Pierre CARTRON. "Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells." Biochemical Journal 356, no. 1 (May 8, 2001): 171–80. http://dx.doi.org/10.1042/bj3560171.
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The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5′ distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.
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Lee, Soohee, Melissa Lin, Aldo Mele, Ying Cao, James Farmar, David Russo, and Colvin Redman. "Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein." Blood 94, no. 4 (August 15, 1999): 1440–50. http://dx.doi.org/10.1182/blood.v94.4.1440.
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Abstract Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.
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Lee, Soohee, Melissa Lin, Aldo Mele, Ying Cao, James Farmar, David Russo, and Colvin Redman. "Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein." Blood 94, no. 4 (August 15, 1999): 1440–50. http://dx.doi.org/10.1182/blood.v94.4.1440.416k01_1440_1450.
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Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.
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Bianchini, Giampaolo, Xiao Qian Wang, Esther Danenberg, Chiun-Sheng Huang, Daniel Egle, Maurizio Callari, Begoña Bermejo, et al. "Abstract GS1-00: Single-cell spatial analysis by imaging mass cytometry and immunotherapy response in triple-negative breast cancer (TNBC) in the NeoTRIPaPDL1 trial." Cancer Research 82, no. 4_Supplement (February 15, 2022): GS1–00—GS1–00. http://dx.doi.org/10.1158/1538-7445.sabcs21-gs1-00.
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Abstract Background Immunecheckpoint inhibitors are effective in early and advanced TNBC, however only aminority of patients benefit making precision immune-oncology a major unmetneed. Imaging mass cytometry (IMC) enables high dimensional tissue imaging atsubcellular resolution for assessment of TNBC ecosystems, providing informationon cell type composition, functional status, and spatial organisation. Methods InNeoTRIP patients with TNBC were randomized to eight cycles ofnab-paclitaxel/carbo (CT) with/without atezolizumab (CTA). Forty-four proteinsspanning cancer cells and the tumor microenvironment (TME) were assessed onpre-treatment biopsies (n=243/280; 86.8% evaluable after QC). FFPE samples werelabelled with antibodies conjugated to isotopically pure rare earth metalreporters and profiled at one micron resolution by IMC. For each sample, wehave generated three high dimensional images that encompass the tumor,tumor-stroma interface and adjacent stroma. We investigated the association ofprotein expression assessed separately for epithelial and TME cells, cellphenotypes, and spatial architectures with PD-L1 status (Ventana SP142),stromal TILs, TNBC types and pathological complete response (pCR). 237 patients(84.6%) have both IMC and RNA-seq available allowing for comparison with genesignatures derived from HALLMARK,ConsensusTME immune cell types, and Nanostring. Results Across243 samples we identify just over one million single cells. By supervised clustering,we defined 37 robust cell phenotypes. PD-L1-positive tumors, high stromal TILsand TNBC type were characterized by extreme heterogeneity and unique cell-type andspatial TME composition. Severalbiomarkers demonstrated a significant test for interaction. Considering proteinexpression, GATA3 and CD20 on TME, HLA-DR in epithelial cells and Ki67 assessedboth in epithelial and TME, had a significant test for interaction (p <0.05). For all these biomarkers, high expression (above median) was associatedwith an increase of pCR of >10% in favour of atezolizumab, whereas lowerexpression group demonstrated a similar pCR rate among arms.Two cellphenotypes, PD-L1+IDO+ antigen presenting cells (APCs) and CD56+ neuroendocrine(NE) epithelial cells, had a significant test for interaction. Higherexpression of these biomarkers was associated with higher likelihood of pCR in CTAarm, but not in CT arm. For example, PD-L1+IDO+APCs in the CTA arm wereassociated with pCR proportions of 64.6% and 24.6% for above- and below-mediangroups respectively (OR4.5 [2.01-10.1], p<0.001).Mostof these tests of interaction retained significance after adjustment by PD-L1status and stromal TILs. Notably, none among 61 gene-expression basedimmune-related pathways and 7 proliferation-related signatures demonstrated a significant test ofinteraction. Resultsof systematic multi-tiered image analysis at the levels of cell-cellinteractions and recurrent higher order multicellular complexes defining TNBC ecosystemsidentified by graph-based methods will be presented at the meeting. Conclusions Imaging mass cytometry provides a morecomprehensive overview of TNBC heterogeneity at a single-cell level withspatial resolution. Bulk protein or gene expression might deliver limitedpredictive information because it does not consider the cell compartment ofexpression. Precise cell phenotyping highlights the predictive role ofPD-L1+IDO+APCs and CD56+NE epithelial cells. Overall, we demonstrated that IMCis feasible in a large, randomized trial and provides independent predictiveinformation on immune checkpoint inhibitors benefit to PD-L1, TILs and gene-expressionprofiles. Citation Format: Giampaolo Bianchini, Xiao Qian Wang, Esther Danenberg, Chiun-Sheng Huang, Daniel Egle, Maurizio Callari, Begoña Bermejo, Claudio Zamagni, Marc Thill, Anton Anton, Matteo Dugo, Stefania Zambelli, Stefania Russo, Eva Maria Ciruelos, Richard Greil, Vladimir Semiglazov, Marco Colleoni, Catherine Kelly, Gabriella Mariani, Lucia Del Mastro, Balázs Győrffy, Olivia Biasi, Pinuccia Valagussa, Giuseppe Viale, Luca Gianni, H Raza Ali. Single-cell spatial analysis by imaging mass cytometry and immunotherapy response in triple-negative breast cancer (TNBC) in the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-00.
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Marsh, WL. "Molecular biology of blood groups: cloning the Kell gene." Transfusion 32, no. 2 (February 1992): 98–101. http://dx.doi.org/10.1046/j.1537-2995.1992.32292180158.x.
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Blavy, G., D. Thiam, and L. Diakhate. "The kell system in Senegal: phenotype and gene frequencies." International Journal of Anthropology 2, no. 1 (March 1987): 75–76. http://dx.doi.org/10.1007/bf02442074.
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Frey, D., M. Machler, R. Seger, W. Schmid, and SH Orkin. "Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: fine mapping of the Xk gene locus." Blood 71, no. 1 (January 1, 1988): 252–55. http://dx.doi.org/10.1182/blood.v71.1.252.252.
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Abstract In a patient suffering from X-linked chronic granulomatous disease (X- CGD)--a disorder of phagocytesuperoxide generation--and McLeod syndrome, characterized by the absence of the red cell Kell antigen, we identified a deletion of the entire X-CGD gene by means of DNA hybridization with a cDNA probe. Our findings suggest that the X-CGD and McLeod loci are physically close in the p21 region of the X chromosome proximal to the Duchenne muscular dystrophy locus.
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Frey, D., M. Machler, R. Seger, W. Schmid, and SH Orkin. "Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: fine mapping of the Xk gene locus." Blood 71, no. 1 (January 1, 1988): 252–55. http://dx.doi.org/10.1182/blood.v71.1.252.bloodjournal711252.
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In a patient suffering from X-linked chronic granulomatous disease (X- CGD)--a disorder of phagocytesuperoxide generation--and McLeod syndrome, characterized by the absence of the red cell Kell antigen, we identified a deletion of the entire X-CGD gene by means of DNA hybridization with a cDNA probe. Our findings suggest that the X-CGD and McLeod loci are physically close in the p21 region of the X chromosome proximal to the Duchenne muscular dystrophy locus.
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Dugo, Matteo, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Robert S. Seitz, Tyler J. Nielsen, et al. "Abstract P2-07-12: Triple negative breast cancer subtypes and early dynamics of the 27-gene IO score predict pCR in the NeoTRIPaPDL1 trial." Cancer Research 82, no. 4_Supplement (February 15, 2022): P2–07–12—P2–07–12. http://dx.doi.org/10.1158/1538-7445.sabcs21-p2-07-12.
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Abstract Background A post-hoc of NeoTRIP trial showed that 27-gene IO score assessed on baseline samples is predictive of increased pathological complete response (pCR) with the addition of atezolizumab to carboplatin/nab-paclitaxel, whereas the LAR subtype has the lowest rate of pCR with and without atezolizumab (Bianchini G ESMO 2021). We evaluated 27-gene IO score and TNBC subtypes on biopsies collected during treatment, assessed biomarker dynamics, and studied the association with pCR. Methods In NeoTRIP, patients randomly received 8 cycles of nab-paclitaxel/carbo alone (CT) or with atezolizumab (CT/A). 258 patients were evaluable for pCR (Per-Protocol Population). We assessed IO score as binary and continuous variable, and the five 101-gene TNBC types (BL1, BL2, LAR, M, and MSL; Ring et al 2016) by RNA-seq on biopsies at baseline and day 1 of second treatment cycle (d1c2) (n: baseline 242/258, 94%; d2c2 161/258, 62%; paired 152/258, 59%). Forty-four paired samples were excluded due to lack of tumor cells at d1c2. PD-L1 (Ventana SP142) and sTILs data were available. We evaluated the association with pCR of biomarkers assessed at d1c2 and their dynamics from baseline. Results Frequency of TNBC types at d1c2 showed minor differences between arms (p = 0.055). TNBC type frequencies were 22.9% BL1, 11.4% BL2, 22.9% LAR, 21.4% M, and 21.4% MSL in the CT/A arm and 43.8% BL1, 6.2% BL2, 11.2% LAR, 21.2% M, and 17.5% MSL in the CT arm. Individual TNBC type changes from baseline to d1c2 were observed, but overall, it was not significant. Frequency of IO positive score at d1c2 was similar in CT and CT/A arm (p = 0.75). Only in CT/A, an increase from baseline to d1c2 was observed (30.9% to 49.3%, p = 0.04).Overall, TNBC types at d1c2 were predictive of pCR (p = 0.00002). Compared to BL1, LAR and M were associated with lower pCR rate in CT (OR = 0.09, 95% CI = 0.01-0.83, p = 0.034 for LAR; OR = 0.16, 95% CI = 0.04-0.66, p = 0.011 for M) and CT/A arm (OR = 0.05, 95% CI = 0.01-0.49, p = 0.010 for LAR; OR = 0.28, 95% CI = 0.06-1.28, p = 0.102). pCR rate in LAR was 11.1% and 6.2% in CT and CT/A arm, respectively. TNBC types were predictive of pCR independently of PD-L1 and sTILs.Continuous IO score at d1c2 was predictive of pCR in both CT/A (p = 0.004) and CT arms (p = 0.009). The binary IO score was significantly associated to higher pCR rate in CT/A arm only (OR = 5.42, 95% CI = 1.95-15.07, p = 0.001). A strong predictive value of the highest quartile of IO score compared to the lowest was observed in CT/A (OR = 14.73, 95% CI = 2.97-73.21, p = 0.001) and CT (OR = 4.38, 95% CI = 1.21-15.81, p = 0.024) arms. pCR rates for the highest and lowest quartiles were 72.2% vs 15.0% in CT/A and 65.2% vs 30.0% in CT arm. In CT/A binary IO score at d1c2 retained significance after adjustment for baseline PD-L1 and sTILs (p = 0.036).Combining baseline and d1c2 IO score, only d1c2 assessment was informative in CT arm. In CT/A arm, both biomarkers were informative, with assessment at d1c2 being more informative than baseline IO score when continuous scores were considered. Baseline binary IO score (OR = 25.0, 95% CI = 3.31-188.9, p = 0.002) and ΔIO score (d1c2-baseline) (OR = 11.3, 95% CI = 1.07-120.1, p = 0.044) retained significance. The combination of baseline and d1c2 binary IO score defined four groups with different likelihood of pCR: 73.7% vs 15.2% in positive/positive and negative/negative groups, respectively (OR = 15.68, 95% CI = 3.88-63.32, p = 0.0001). Conclusions Dynamic of IO score early on treatment was linked to likelihood of pCR independently of baseline biomarkers and may be an early surrogate of treatment benefit especially in atezolizumab arm. LAR and M are associated with lower pCR rate, suggesting that different therapeutic strategies may be beneficial. Combining baseline and on-treatment biomarkers can be more informative than baseline only of the complex tumor/immune co-evolution dynamic and of clinical outcome. Citation Format: Matteo Dugo, Chiun-Sheng Huang, Daniel Egle, Begoña Bermejo, Claudio Zamagni, Robert S. Seitz, Tyler J. Nielsen, Marc Thill, Antonio Anton, Stefania Russo, Eva Maria Ciruelos, Brock L. Schweitzer, Douglas T. Ross, Barbara Galbardi, Richard Greil, Vladimir Semiglazov, Balázs Gyorffy, Marco Colleoni, Catherine Kelly, Gabriella Mariani, Lucia Del Mastro, Pinuccia Valagussa, Giuseppe Viale, Maurizio Callari, Luca Gianni, Giampaolo Bianchini. Triple negative breast cancer subtypes and early dynamics of the 27-gene IO score predict pCR in the NeoTRIPaPDL1 trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-12.
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Lee, S., E. Zambas, ED Green, and C. Redman. "Organization of the gene encoding the human Kell blood group protein [published erratum appears in Blood 1996 Jun 1;87(11):4922]." Blood 85, no. 5 (March 1, 1995): 1364–70. http://dx.doi.org/10.1182/blood.v85.5.1364.bloodjournal8551364.
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Kell is one of the major blood group systems in human erythrocytes. It is a complex system containing a large number of different antigens. Previously we cloned the Kell cDNA, which was predicted to encode an integral membrane protein with 731 amino acids. Now we have isolated overlapping genomic clones and determined the exon-intron structure of the KEL gene; it spans approximately 21.5 kb with its coding sequence being organized in 19 exons that range in size from 63 bp to 288 bp. The size of introns ranges from 93 bp to approximately 6 kb. The donor and acceptor splice sites all conform to the consensus splicing sequences. Exon 1 encodes only the initiation amino acid, methionine, and contains a consensus Sp1 binding site. The single membrane spanning region of Kell protein is encoded in exon 3 and the putative zinc endopeptidase active site is in exon 16. The amino acids encoded by the 19 exons are identical to those of a person with a common Kell phenotype, as determined by RNA polymerase chain reaction of peripheral blood. Amplification of cDNA 552 ends, derived from human fetal liver, indicated three transcription initiation sites located 30, 81, and 120 bp upstream of the initiation codon. The 552 flanking region of KEL from -176 does not contain a TATA sequence, but has possible GATA-1 binding sites and has significant promoter activity when determined by chloramphenicol acetyltransferase activity in K562 cells.
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Visconte, Valeria, Bartlomiej P. Przychodzen, Steffan T. Nawrocki, Swapna Thota, Kevin R. Kelly, Bhumika Patel, Cassandra M. Hirsch, et al. "Genetic and Epigenetic Defects in the Autophagy Machinery in Myelodysplastic Syndromes." Blood 128, no. 22 (December 2, 2016): 4301. http://dx.doi.org/10.1182/blood.v128.22.4301.4301.
Full textAbstract:
Abstract Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202). We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off >20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1K700E compared to SF3B1WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1K700E patients showed increased autophagosomes compared to SF3B1WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08). In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.