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Articles de revues sur le sujet "SET-2 cell lines"
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Santamaria, P., T. Utsugi, B. J. Park, N. Averill, S. Kawazu et J. W. Yoon. « Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences. » Journal of Immunology 154, no 5 (1 mars 1995) : 2494–503. http://dx.doi.org/10.4049/jimmunol.154.5.2494.
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Abstract Insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a cell-mediated autoimmune process against pancreatic beta-cells. We have shown that beta-cell-cytotoxic CD8+ T cell clones can transfer IDDM to irradiated NOD mice if co-injected with nondiabetogenic CD4+ spleen T cells. To determine whether CTLs recruited to pancreatic islets recognize a restricted set of local Ags, we sequenced TCR-alpha and TCR-beta cDNA generated by anchor PCR from CD8+ CTL lines and clones derived from islets of 10 different NOD mice. These CTL lines were oligoclonal, but did not show skewed V alpha, V beta, J alpha, or J beta gene usage when compared with CD8+ spleen T cells. However, of the 26 different CTL-derived TCR-alpha sequences from all of these CTL lines and clones, 17 (65%) used one of three highly related, N region-encoded, CDR3 motifs. Motifs 1 and 2 (7 clonotypes each) contained a hydrophobic amino acid followed by Arg and a negatively charged or a polar residue (Asn or Gly), respectively. Motif 3 (3 clonotypes) was x-Arg-Gly. In 12 of these 17 rearrangements, the core sequence was followed by Tyr or Ser. By contrast, none of 31 different TCR-alpha rearrangements used by CD8+ spleen T cells encoded motifs 1 or 2, and only one encoded motif 3. Different TCR-beta rearrangements within individual lines also used homologous CDR3 sequences, but these sequences varied between lines. Skewed TCR-alpha-CDR3 usage by islet-derived CTLs was substantiated further by isolation of CTL clones transcribing highly homologous TCR-alpha, but different TCR-beta, rearrangements. These data suggest that CTLs recruited to pancreatic islets during spontaneous IDDM recognize a restricted set of beta-cell autoantigenic determinants.
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Quentmeier, Hilmar, Claudia Pommerenke et Hans G. Drexler. « Epigenetic Modifier Mutations in the LL-100 Panel ». Blood 132, Supplement 1 (29 novembre 2018) : 5271. http://dx.doi.org/10.1182/blood-2018-99-110060.
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Abstract The NCI-60 human cell line panel, developed for use in drug development comprises sixty human cancer cell lines derived from nine different tissues. Only six cell lines of the NCI-60 were derived from blood cancers. Therefore, most forms and subtypes of leukemia and lymphoma are not represented in the NCI-60 panel. To respond to this apparent gap, we suggest the novel LL-100 panel, 100 leukemia and lymphoma cell lines representing the major leukemia/lymphoma entities, for basic research and drug development. Whole exome sequencing and RNA sequencing were performed to identify mutations in 100 cell lines. Here we list the 100 cell lines, ordered by subtype and show mutations in epigenetic modifier genes. We found cell lines with mutations in ASXL1, EZH2, IDH1, TET2 and in DNMT3A. Hitherto, cell line OCI-AML3 was the only human cell line described with a DNMT3A mutation. Twenty-two percent of patients with acute myeloid leukemia contain DNMT3A mutations and the median overall survival with DNMT3A mutations is shorter than without. Most DNMT3A mutations are heterozygous and alter amino acid R882, R882H being the most common DNMT3A mutation in AML. Exogenously mutant murine R878H (equivalent to human R882H) inhibits DNMT3A activity in a dominant negative manner. We describe here that the AML cell line SET-2 carries a heterozygous G to A transition at chr2_25234373 (hg38) which leads to the DNMT3A R882H amino acid substitution. Chip-based methylation analysis revealed that the described DNMT3A targets IRF8, KLF2, HOXA11 and HOXB2 are hypomethylated in cell lines OCI-AML3 (DNMT3A R882C) and in SET-2 (DNMT3A R882H). These data suggest that SET-2 is a novel model cell line for functional analysis of the DNMT3A R882 mutation and a first gain in knowledge through data mining the LL-100 panel. Disclosures No relevant conflicts of interest to declare.
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Moudgil, Tarsem, Bernard Fox et Hong-Ming Hu. « 85 Detection of human angiotensin-converting enzyme 2 receptor (hACE2R) on human cancer cell lines ». Journal for ImmunoTherapy of Cancer 9, Suppl 2 (novembre 2021) : A93. http://dx.doi.org/10.1136/jitc-2021-sitc2021.085.
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BackgroundSARS-CoV-2 infections have delayed administration of treatments for some patients with cancer, increasing the number of avoidable deaths. However, we hypothesized that infection of cancer cells with SARS-CoV-2 might increase the immunogenicity of those cancer cells. Here we sought to determine whether non-small cell lung cancer (NSCLC) and head and neck squamous cell cancer (HNSCC) cell lines could be a potential target of SARS-CoV-2, which binds and infects host cells via interactions between the viral spike glycoprotein and the human angiotensin-converting enzyme 2 receptor (hACE2) receptor. Through an institutional research tissue protocol, our lab has established a panel of cancer cell lines of various histologies. Here we set out to identify whether HNSCC and NSCLC cell lines expressed the hACE2R. We also investigated the expression of neuropilin-1, a molecule reported to facilitate SARS-CoV-2 cell entry.MethodsEstablished cell lines were phenotyped by flow cytometric analysis utilizing the anti-hACE2R antibody from Novus Biologicals. Cell lines were also stained with mIgG1 and anti-CD3 antibodies as negative staining controls.ResultsWe identified that three of eight NSCLC cell lines expressed the hACE2R and two of these had strong expression of neuropilin-1. Evaluation of HNSCC cell lines identified seven of seven cell lines expressed detectable levels of hACE2R but only one of seven HNSCC celllines expressed substantial levels of neuropilin-1. Preliminary evaluation of a renal cell carcinoma (RCC) cell line revealed strong staining for hACE2R.ConclusionsOur study found that a majority of HNSCC cell lines (100% n=7) and approximately a third of the NSCLC cell lines (37.5%, n=8) tested express the hACE2R. Some cell lines express both hACE2R and neuropilin-1, potentially increasing their susceptibility for infection with SARS-C0V-2. While these studies were performed with cultured cell lines that may have modulated expression of hACE2R, it is possible that in vivo these tumors express the ACE2R and could serve as a target and possible reservoir for SARS-CoV-2.AcknowledgementsSupportThe Chiles foundation, Nancy Lematta
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Nagai, T., H. Harigae, H. Ishihara, H. Motohashi, N. Minegishi, S. Tsuchiya, N. Hayashi, L. Gu, B. Andres et JD Engel. « Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines ». Blood 84, no 4 (15 août 1994) : 1074–84. http://dx.doi.org/10.1182/blood.v84.4.1074.1074.
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Abstract To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.
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Nagai, T., H. Harigae, H. Ishihara, H. Motohashi, N. Minegishi, S. Tsuchiya, N. Hayashi, L. Gu, B. Andres et JD Engel. « Transcription factor GATA-2 is expressed in erythroid, early myeloid, and CD34+ human leukemia-derived cell lines ». Blood 84, no 4 (15 août 1994) : 1074–84. http://dx.doi.org/10.1182/blood.v84.4.1074.bloodjournal8441074.
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To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.
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Resar, Linda, Donna Marie Williams, Lingling Xian, Wenyan Lu, Briyana Chisholm, Li Luo, Zhizhuang Joe Zhao, Ophelia Rogers, Jerry L. Spivak et Alison R. Moliterno. « High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models ». Blood 132, Supplement 1 (29 novembre 2018) : 102. http://dx.doi.org/10.1182/blood-2018-99-119549.
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Abstract Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression. Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis. Results: HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. Disclosures No relevant conflicts of interest to declare.
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Lindhout, E., A. Lakeman, M. L. Mevissen et C. de Groot. « Functionally active Epstein-Barr virus-transformed follicular dendritic cell-like cell lines. » Journal of Experimental Medicine 179, no 4 (1 avril 1994) : 1173–84. http://dx.doi.org/10.1084/jem.179.4.1173.
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Follicular dendritic cells (FDC) are unique nonlymphoid cells found only in germinal centers. FDC can be distinguished from other accessory cells based on a characteristic set of cell surface markers. It is known that FDC are able to rescue germinal center B cells from apoptosis. To investigate the role of FDC in the process of selection and maturation of B cells during germinal center reactions, we tried to establish factor-independent immortalized FDC-like cell lines. Because freshly isolated FDC express the Epstein-Barr Virus (EBV) receptor CD21, we attempted EBV transformation on isolated FDC. After incubation of FDC-enriched cell populations with EBV, cell lines were obtained consisting of slowly duplicating very large cells. These cell lines have a fibroblast-like morphology but could be clearly distinguished from several human fibroblast cell lines by displaying a different phenotype including intercellular adhesion molecule 1, CD40, and CD75 expression. Detection of the EBV-encoded proteins latent membrane protein 1 and Epstein-Barr virus nuclear antigen 2 in our FDC-like cell lines implicated successful EBV transformation. FDC-like cells are able to bind nonautologous B cells and preserve the latter from apoptosis. The binding of B cells to FDC-like cells is dependent on adhesion via lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and closely resembles the pattern of emperipolesis as described by others. These data demonstrate that FDC can be successfully infected by EBV, and that the cell lines obtained share phenotypic and functional characteristics with freshly isolated FDC.
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Troeger, Anja, Pascal-David Johann, Mumine Senturk, Michael D. Milsom et David A. Williams. « Intact Rac Signaling Is Important for Leukemia Cell Survival ». Blood 116, no 21 (19 novembre 2010) : 2885. http://dx.doi.org/10.1182/blood.v116.21.2885.2885.
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Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.
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Verheul, C., T. V. Kers, A. Van Der Ploeg, M. Van Der Kaaij, M. Aghababazadeh, M. De Wit, Y. Hoogstrate et al. « P11.47 Generation, characterisation and drug screening of patient-derivedIDH1-mutated glioma cell lines ». Neuro-Oncology 21, Supplement_3 (août 2019) : iii54. http://dx.doi.org/10.1093/neuonc/noz126.193.
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Abstract BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.
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Gozgit, Joseph M., Geraldine A. Bebernitz, Pankaj Patil, Minwei Ye, Jiaquan Wu, Stephanos Ioannidis, Audrey Davies, Tao Wang, Dennis Huszar et Michael Zinda. « Effects of a Novel, Selective Jak2 Inhibitor, AZ60, on STAT5 Signaling and Cellular Growth in Jak2 V617F Cell Lines. » Blood 110, no 11 (16 novembre 2007) : 3549. http://dx.doi.org/10.1182/blood.v110.11.3549.3549.
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Abstract A role for Jak2 in the etiology of the myeloproliferative diseases (MPDs) was discovered with the identification of a single activating point mutation, V617F, in the pseudokinase domain of JAK2. We have developed a Jak2 inhibitor, AZ60, which inhibits in vitro JAK2 enzyme activity with a Ki of 0.45 nM. AZ60 demonstrates inhibition of STAT5 phosphorylation and proliferation in a Tel-Jak2 engineered cell line with IC50 values of 18 and 23 nM, respectively. To understand the selectivity versus other Jak kinase family members we engineered three additional cell lines containing Tel fusions with the kinase domains of Jak1, Jak3 and Tyk2. Under these settings, AZ60 demonstrates a 15 to 30-fold selectivity for Tel-Jak2 driven STAT5 phosphorylation when compared to other Jak kinase family members. AZ60 was also tested for its ability to inhibit STAT5 phosphorylation and cellular proliferation in two human hematological cell lines, Set-2 and Hel. Set-2 expresses both wt and V617F Jak2, while Hel is homozygous for the Jak2 V617F mutation. AZ60 decreased phospho-STAT5 levels in a dose-dependent manner in both Set-2 and Hel cells with IC50 values of 15 and 25 nM, respectively. Complete inhibition of proliferation and a marked induction of apoptosis were observed in both cell lines following treatment with AZ60. Induction of apoptosis by AZ60 was characterized by a time- and dose-dependent increase in caspase 3/7 activities and PARP-cleavage. These data demonstrate AZ60 is a potent and selective inhibitor of Jak2 and may help decipher the mechanisms underlying Jak2-driven myeloproliferative disease.
Thèses sur le sujet "SET-2 cell lines"
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Marino, Luigi. « Ruolo del Ruxolitinib nel cross-talk tra cellule staminali mesenchimali e microambiente midollare nella mielofibrosi ». Doctoral thesis, Universita degli studi di Salerno, 2019. http://elea.unisa.it:8080/xmlui/handle/10556/4243.
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2017 - 2018Introduction; Mesenchymal stem cells (Mesenchymal Stem Cells, MSC) are one of the most studied and well-characterized adult stem cell populations. They are excellent candidates in regenerative medicine, mainly because of their immunomodulatory properties and their emerging role in intercellular communication. MSCs as cellular components of bone marrow hematopoietic niche play a fundamental role in maintaining the physiological balance of the niche and in promoting and regulating hematopoietic stem cell (HSCs) functions, such as proliferation and "homing" to the bone marrow. - Methods: In this work, we focused on the possible internalization and release of Ruxolitinib (a JAK1/2 inhibitor by NOVARTIS Pharma) by MSC. In details, primary human MSC were isolated from bone marrow (BMMSC) of five patients diagnosed with Idiopathic Myelofibrosis or Polycythemia Vera. Diagnosis was confirmed by histopathology and molecular biology for the detection of mutations in Janus Kinase 2 receptor encoding gene, specifically for JAK2 V617F mutation. Subsequently, we evaluated the in vitro anti-proliferative effect of culture medium conditioned with Ruxolitinib on immortalized JAK2+ CD34+ SET-2 cells. Finally, a co-culture system of BMMSC and SET-2 cells treated or not with Ruxolitinib in different ratios (1:20, 1: 100 and 1: 1000) was used for estimating the relative anti-proliferative action on SET-2 cell line. - Results: Our preliminary results showed that MSCs could uptake and release Ruxolitinib in culture medium, and conditioned culture medium had more anti-proliferative effects on SET-2 cells compared to the drug alone added to the medium. In an in vitro co-culture system, the proliferation of SET-2 cells decreased by increasing MSC ratio treated with Ruxolitinib / SET-2, and BMMSC treated with Ruxolitinib had a greater anti-proliferative action on SET-2 cells compared to untreated BMMSCs. - Conclusions: Mesenchymal bone marrow stem cells could uptake and release Ruxolitinib that might increase the anti-proliferative effect of the drug on SET-2 cell line carrying the JAK2 V617F mutation. These mechanisms may contribute to amplify over time the pharmacological effects of Ruxolitinib in the bone marrow niche of Idiopathic Myelofibrosis and Polycythemia Vera patients. [edited by Author]
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Chapitres de livres sur le sujet "SET-2 cell lines"
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Taber, Douglass F. « The Reisman Synthesis of (+)-Salvileucalin B ». Dans Organic Synthesis. Oxford University Press, 2015. http://dx.doi.org/10.1093/oso/9780190200794.003.0084.
Texte intégralRésumé :
Salvileucalin B 3 exhibits modest cytotoxicity against A549 (human lung adenocarcinoma) and HT-29 (human colon adenocarcinoma) cell lines. The compelling interest of 3 is its complex, highly functionalized heptacyclic skeleton. Sarah E. Reisman of the California Institute of Technology envisioned (J. Am. Chem. Soc. 2011, 133, 774) that the intramolecular cyclization of the diazo ketone 1 could offer an efficient entry to 2 and thus to 3. The key intermediate for the preparation of 1 was the acid 11. It was not possible to achieve communication between the two stereogenic centers of 11, so the decision was taken to establish these independently. This led to a strategy centered on the construction of the 1,2,3,4-tetrasubstituted aromatic ring. The absolute configuration of the stereogenic center of 8 was set by enantioselective addition of 5 to the commercial aldehyde 4. The absolute configuration of the second center was set using the Myers protocol. Although 10 could not be hydrolyzed without epimerization, cyclization followed by hydrolysis was effective, delivering 11 as a 10:1 mixture of diastereomers. From the algebra of mutual resolution, the major diastereomer, separated at a later stage, was of high enantiomeric purity. The acid 11 was homologated, first by the Arndt-Eistert procedure, then by condensation of the methyl ester so prepared with the anion of acetonitrile. Exposure of the derived diazo nitrile 1 to Cu catalysis under brief microwave irradiation led to smooth cyclization to the hexacyclic ketone 2. Although the skeleton of 2 was readily assembled, it is highly strained. This was made clear on Dibal reduction of the derived enol triflate. The product was clearly not the desired aldehyde 2, but rather 12, the product of Claisen rearrangement. Reasoning that the Claisen rearrangement is thermally reversible, and that the ether 12 would be stable to hydride, they carried forward with Dibal reduction—and were rewarded by the appearance of the desired primary alcohol from the reduction of 2. Pd-mediated cyclocarbonylation delivered 13, which was selectively oxidized to (+)-salvileucalin B 3.
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Taber, Douglass F. « The Shair Synthesis of Cephalostatin 1 ». Dans Organic Synthesis. Oxford University Press, 2013. http://dx.doi.org/10.1093/oso/9780199965724.003.0093.
Texte intégralRésumé :
The cephalostatins and ritterazines, represented by cephalostatin 1 3, have the remarkable property of inducing apoptosis in apoptosis-resistant malignant cell lines. The total synthesis ( J. Am. Chem. Soc. 2010, 132, 275) of 3 by Matthew D. Shair of Harvard University required the practical preparation of the complex hexacyclic ketones 1 and 2. The preparation of 1 started with irradiation of commercial hecogenin acetate 4 to give the known aldehyde 5 . Reaction of 5 with N -phenyltriazolenedione 6 led to the ketal 7. Oxidative cleavage generated an aldehyde, which on reduction and allylation was converted to 8. Acid-mediated cyclization led to 9. The sidechain of 9 was removed, giving 10, which was selectively reduced, leading to 11. Intramolecular aldol condensation gave 12. The relative configuration of the spiroketal 1 was established by kinetic bromoetherification of the alcohol 13, followed by free radical reduction of the resulting tertiary bromide, and acid-catalyzed equilibration. The synthesis of 2 began with the inexpensive steroid 14. Following the Schönecker protocol, C-H functionalization led to the ketone 15. Pd-mediated coupling of the derived enol triflate with the alkyne gave 16, which was oxidized and cyclized to 17. Simmons-Smith conditions converted the dihydrofuran of 17 into the cyclopropane, which was again opened kinetically with Br (NBS) to set the relative configuration of the spiroketal. Free radical reduction followed by protection and oxidation then completed the preparation of 2. The coupling of 1 and 2 (not illustrated) to form the central pyrazine of 3 followed the precedent of Fuchs, combining the 2-azido ketone derived from 1 with the 2-amino methoxime derived from 2. Remarkably, tens of milligrams of 1 and of 2 were prepared, assuring a reasonable supply of 3 for further studies.
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Taber, Douglass F. « The Nakada Synthesis of (+)-Ophiobolin A ». Dans Organic Synthesis. Oxford University Press, 2017. http://dx.doi.org/10.1093/oso/9780190646165.003.0084.
Texte intégralRésumé :
Ophiobolin A 3 shows nanomolar toxicity toward a range of cancer cell lines. A central feature of this sesterterpene, isolated from the rice fungus Ophiobolus miyabeanus, is the highly-substituted eight-membered ring. A key step in the synthesis of 3 described (Chem. Eur. J. 2013, 19, 5476; Angew. Chem. Int. Ed. 2011, 50, 9452) by Masahisa Nakada of Waseda University was the acid-mediated cyclization of 1 to 2. The preparation of 1 began with the enantioselective hydrolysis of 4 to the monoester 5. Selective reduction followed by protection gave 6, that was carried on via 7 to 8. The ethoxyethyl group was selectively removed, and the alcohol was converted to an iodide (not illustrated) that was condensed with the lactone 9 to give 1. The cyclization of 1 could jeopardize the stereogenic center adjacent to the masked carbonyl, so eight diastereomers were possible. Careful optimization led to a preparatively useful yield of the desired product 2. Hydroboration gave 10, that was carried on to the aldehyde 11. The cyclopentanone 15 was prepared from the enantiomerically-enriched epoxide 12. Opening with vinyl magnesium bromide followed by exposure to the second-generation Grubbs catalyst gave the diol 13, that was selectively protected, leading to 14. The derived bromohydrin was a mixture of regioisomers and diastereomers, from which, after oxidation, 15 dominated. Generation of the boron enolate from 15 in the presence of 11 gave the aldol product, that could be dehydrated with the Burgess reagent. Reduction with Raney nickel set the stereogenic center adjacent to the ketone, that was carried on to 16. Metathesis to close the eight-membered ring was not trivial. Finally, it was found that 17 could be induced to cyclize to 18 at an elevated temperature using the second-generation Hoyveda catalyst. Protecting group exchange gave 19. Routine functional group manipulation then completed the synthesis of (+)-ophiobolin 3. Some years ago, Neil E. Schore of the University of California, Davis showed (Tetrahedron Lett. 1994, 35, 1153) that the opening of Sharpless-derived epoxides such as 12 with vinyl nucleophiles was unexpectedly flexible. One set of conditions gave the expected inversion, but alternative conditions led to opening with clean retention (or double inversion) of absolute configuration.
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Mclntyre, Donald B. « An Executable Notation, With Illustrations From Elementary Crystallography ». Dans Computers in Geology - 25 Years of Progress. Oxford University Press, 1994. http://dx.doi.org/10.1093/oso/9780195085938.003.0024.
Texte intégralRésumé :
Elementary crystallography is an ideal context for introducing students to mathematical geology. Students meet crystallography early because rocks are made of crystalline minerals. Moreover, morphological crystallography is largely the study of lines and planes in real three-dimensional space, and visualizing the relationships is excellent training for other aspects of geology; many algorithms learned in crystallography (e.g., rotation of arrays) apply also to structural geology and plate tectonics. Sets of lines and planes should be treated as entities, and crystallography is an ideal environment for introducing what Sylvester (1884) called "Universal Algebra or the Algebra of multiple quantity." In modern terminology, we need SIMD (Single Instruction, Multiple Data) or even MIMD. This approach, initiated by W.H. Bond in 1946, dispels the mysticism unnecessarily associated with Miller indices and the reciprocal lattice; edges and face-normals are vectors in the same space. The growth of mathematical notation has been haphazard, new symbols often being introduced before the full significance of the functions they represent had been understood (Cajori, 1951; Mclntyre, 1991b). Iverson introduced a consistent notation in 1960 (e.g., Iverson 1960, 1962, 1980). His language, greatly extended in the executable form called J (Iverson, 1993), is used here. For information on its availability as shareware, see the Appendix. Publications suitable as tutorials in , J are available (e.g., Iverson. 1991; Mclntyre, 1991 a, b; 1992a,b,c; 1993). Crystals are periodic structures consisting of unit cells (parallelepipeds) repeated by translation along axes parallel to the cell edges. These edges define the crystallographic axes. In a crystal of cubic symmetry they are orthogonal and equal in length (Cartesian). Those of a triclinic crystal, on the other hand, are unequal in length and not at right angles. The triclinic system is the general case; others are special cases. The formal description of a crystal gives prominent place to the lengths of the axes (a, b, and c) and the interaxial angles ( α, β, and γ). A canonical form groups these values into a 2 x 3 table (matrix), the first row being the lengths and the second the angles.
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Cook, Matthew. « Still Life Theory ». Dans New Constructions in Cellular Automata. Oxford University Press, 2003. http://dx.doi.org/10.1093/oso/9780195137170.003.0008.
Texte intégralRésumé :
In Conway’s Game of Life [2], if one starts with a large array of randomly set cells, then after around twenty thousand generations one will see that all motion has died down, and only stationary objects of low period remain, providing a final density of about .0287. No methods are known for proving rigorously that this behavior should occur, but it is reliably observed in simulations. This brings up several interesting related questions. Why does this “freezing” occur? After everything has frozen, what is the remaining debris composed of? Is there some construction that can “eat through” the debris? If we start with an infinitely large random grid, so that all constructions appear somewhere, what will the long term behavior be? It seems clear that knowing the composition of typical debris is central to many such questions. Much effort has gone into analyzing the objects that occur in such stationary debris, as well as into determining what stationary objects can exist at all in Life [4, 8], Both of these endeavors depend on having some notion of what an “object” is in the first place. One simple notion is that of an island, a maximal set of live cells connected to each other by paths of purely live cells. But many common objects, such as the “aircraft carrier,” are not connected so strongly. They are composed of more than one island, but we think of them as a single object anyway, since their constituent islands are not separately stable. Any pattern that is stable (has period one, i.e., does not change over time) is called a still life. Since a collection of stable objects can satisfy this definition, the term strict still life is used to refer to a single indivisible stable object, and pseudo still life is used to refer to a stable pattern that is composed of distinct strict still lifes. For example, the bi-block is a pseudo still life, since it is composed of two blocks, but the aircraft carrier is a strict still life, since its islands are not stable on their own.
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Wong, Agnes. « Ocular Motor Disorders Caused by Lesions in the Brainstem ». Dans Eye Movement Disorders. Oxford University Press, 2008. http://dx.doi.org/10.1093/oso/9780195324266.003.0017.
Texte intégralRésumé :
Oculopalatal tremor usually occurs many months after an initial insult, due to neural deafferentation. It rarely resolves spontaneously. Treatment is with gabapentin, ceruletide, or anticolinergic agents. The y-group is a small group of cells that lies rostral to the inferior cerebellar peduncle. It receives inputs from the saccule (part of the otolith) and from Purkinje cells of the flocculus, and it projects to the oculomotor and trochlear nuclei via the superior cerebellar peduncle and a crossing ventral tegmental tract. 1. Discharge during upward smooth pursuit, optokinetic, and combined eye-head tracking (VOR cancellation), but not during VOR in darkness (see sections 3.14 and 5.2) 2. Together with the flocculus and ventral paraflocculus, may contribute to vertical VOR adaptation (see section 3.12) No known documented clinical correlate Collections of neurons scattered along the midline fiber tracts in the pons and medulla, including: 1. The nucleus pararaphales in the medulla, which receives vertical eye position signals from the INC, and projects to the flocculus and ventral paraflocculus 2. The nucleus incertus in the pons, which contains burst-tonic neurons that mainly discharge in relation to horizontal eye movements, and projects to the flocculus Function: may send an “efference copy” of eye movement commands to the flocculus for gaze holding or longer term adaptation No known documented clinical correlate The abducens nucleus contains: 1. Abducens motoneurons that innervate the ipsilateral lateral rectus 2. Abducens internuclear neurons, the axons of which cross the midline and ascend via the MLF to innervate the contralateral medial rectus motoneurons in the oculomotor nucleus. 3. Neurons that project to the cerebeller flocculus The abducens nucleus is the final common motor pathway for horizontal conjugate eye movements, as it receives input for horizontal saccades, VOR, and smooth pursuit. The paramedian pontine reticular formation (PPRF) contains: Excitatory burst neurons (EBN) in the dorsomedial nucleus reticularis pontis caudalis (NRPC) that ■ Project to the ipsilateral abducens nucleus to generate ipsilateral, conjugate, horizontal saccades ■ Project to inhibitory burst neurons in the nucleus paragigantocellularis dorsalis (PGD) and receive inhibitory inputs from omnipause neurons in nucleus raphe interpositus (rip)
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« designation will be used for the 12E7 antigen. CD99 was first detected by 12E7, a monoclonal antibody made in response to a T-cell line, and was initially thought to be a ‘thymus-leukaemia’ marker antigen [41]. Many similar antibodies were made which reacted with different epitopes of the same molecule [see 42]. Independently, CD99 was identified as E2, a T-cell adhesion molecule, and as a marker antigen for Ewing’s tumours [see 40]. CD99 is expressed on many tissues including red cells. By somatic cell hybridization and biochemical studies, Goodfellow and his colleagues have shown that MIC2, the structural locus encoding the 12E7 antigen, is located on the short arm of the X chromosome and on the short arm of the Y chromosome within the pairing regions [43]. MIC2 has been cloned [44]. XG is X-borne. On red cells, CD99 expression is a quantitative polymorphism [45]. Family studies proved that this polymorphism is also caused by regulator genes on X and Y chromosomes. XG appears to be the regulator on the X [46]. There is variation in CD99 expression on cells other than red cells. In a recent publication, CD99 was found on all haemopoeitic cells but was variably expressed during leucocyte differentiation [40]. Use of different monoclonal antibodies and variability of expression during maturation offered an explanation for the previous apparently contradictory findings by different laboratories. Both Xga and CD99 are sialoglycoproteins [47,48,49]. These glycoproteins differ in Mr and in their sialic acid content [49]. Immunostaining of separated membrane components with 12E7 and similar antibodies had demonstated that the MIC2 gene product was a 30-32 kD protein. 12E7 also bound to an intracellular band of 28 kD which was found in mouse cell lines in addition to human cell lines, platelets, lymphocytes and red cells but it was not encoded by the MIC2 gene [47]. Immunoblotting assays have shown that Xga was associated with two diffuse bands of 22-25 kD and 26.5-29 kD [49]. These findings supported the evidence that Xga and CD99 were products of different structural loci. However, XG appears to regulate CD99 expression on red cells and Latron and colleagues found that purified CD99 protein inhibited binding of 12E7 and of anti-Xga to red cells [48]. We have studied the immunochemical relationship of Xga and CD99 [50]. One approach was immunoprecipitation of membrane components from biotin labelled cells. Bands are detected by chemiluminescence via peroxidase-conjugated avidin. The 32 kD protein of CD99 was visualised by this technique and the quantitative polymorphism was also demonstrated since the 32 kD band is seen on X-ray film after 2 minutes in membranes ». Dans Transfusion Immunology and Medicine, 197. CRC Press, 1995. http://dx.doi.org/10.1201/9781482273441-15.
Texte intégralActes de conférences sur le sujet "SET-2 cell lines"
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Inufusa, H., N. Sagara, K. Nakano et M. Yasutomi. « CHARACTERISATION OF PLATELET AGGREGATING MATERIAL EXTRACTED FROM HUMAN LUNG ADENOCARCINOMA CELL LINE WHICH METASTASIZED IN NUDE MICE S,C, IMPLANTATION ». Dans XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643199.
Texte intégralRésumé :
It has been reported in animal experimental system that Platelet Aggregating Material (PAM) of cancer cell play important role in cancer metastasis. Many human cancer cell lines has been also studied the platelet aggregation activity of PAM. But correlation between the platelet aggregation activity and metastatic potential of human cancer cells were usually unkncwn. We established a human lung adenocarcinoma cell line KUM-LK-2 which produce spontaneous lung metastasis when cells implanted into subcutaneous of nude mice. PAM was extracted from KUM-LK-2 cells following the method of D.Mbhanty and P.Hilgard. Platelet aggregation study of PAM was performed by human Heparinized platelet rich plasma useing NIKO Hematracer PACT2D. Character of PAM were examined by physical and chemical treatment. KUM-LK-2 PAM shew 80% of platelet aggregation in maximum after 150 sec lag time, and aggregation was not found by Citrated PRP.It is concluded that PAM is high moleculer protein and contain Phospholipid and aggregation activity is concerning with ATP composition of platelet. PAM dose not contain Fibrinogen and Sialic acid, and not concern with Throrriboxan composition. This study is first case of platelet aggregation activity of PAM extracted human canoer cells which contain metastatic potential.
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Rodrigues, Júlia Morales, et Camila Biberg. « SIGNIFICADO CLÍNICO DE “HARLEQUIN CELL” NA SÍNDROME HIPEREOSINOFÍLICA PRIMARIA : ESTUDO DE REVISÃO ». Dans II Congresso Brasileiro de Saúde On-line. Revista Multidisciplinar em Saúde, 2021. http://dx.doi.org/10.51161/rems/1513.
Texte intégralRésumé :
Introdução: O sangue é dividido em três partes: serie branca, serie vermelha e plasma. A serie branca é fundamental para o sistema imunológico humano, pois combate substâncias estranhas ao organismo, sendo dividida em 2 grupos: granulócitos (neutrófilos, eosinófilos e basófilos) e agranulócitos (monócitos e linfócitos). Em especial neste trabalho daremos ênfase aos eosinófilos, células granuladas responsáveis principalmente por reações alérgicas e parasitarias; porém, em alguns casos podendo estar relacionados com quadros graves de síndrome hipereosinofílica, onde os eosinófilos se apresentam elevados no esfregaço sanguíneo do paciente (Valor relativo: ≥ 15%). Esta produção aumentada de eosinófilos pela medula óssea acaba resultando em liberação de células displásicas (“Harlequin Cell”) na corrente sanguínea, podendo ser indicativo de neoplasias. Objetivo: Realizar estudo de revisão sobre o significado clínico da presença de eosinófilos displásicos em esfregaço sanguíneo dos pacientes com síndrome hipereosinofílica. Metodologia: Revisão bibliográfica realizada com base em artigos disponíveis no Pubmed de característica online e gratuito dos anos de 2015 a 2021, utilizando os seguintes descritores: eosinófilos displásicos; síndrome hipereosinofílica; leucemia eosinofílica. Resultados: A síndrome hipereosinofílica primaria também conhecida como neoplásica, relaciona o aumento de eosinófilos no sangue periférico com uma anormalidade clonal dos mesmos na medula óssea (mutação genética), sendo alusivo ao aparecimento de eosinófilos displásicos com granulações roxo-violeta e tamanho aumentado (grânulos metacromáticos), esses eosinófilos com morfologia desconforme são conhecidos na hematologia como “Harlequin Cell”. O aparecimento dessas células no hemograma de pacientes com síndrome hipereosinofília primaria em conjunto com sintomas apresentados pode ser um indicativo precoce de uma leucemia mielóide aguda (LMA) ou leucemia eosinofílica, sendo assim é importante observar e relatar esse tipo de anomalia visualizada no esfregaço sanguíneo do paciente. Conclusão: Apesar do estudo sobre o “Harlequin Cell” ser muito escasso devido ao seu baixo número de casos, se faz necessário mais estudos e investigações clínicos/laboratoriais, pois o mesmo pode ser um forte indício prévio de neoplasias hematológicas.
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Dunkers, Joy P., Stefan D. Leigh, Marcus T. Cicerone, Forrest A. Landis, Francis W. Wang et John A. Tesk. « NIST Development of Reference Material Scaffolds for Tissue Engineering ». Dans ASME 2005 International Mechanical Engineering Congress and Exposition. ASMEDC, 2005. http://dx.doi.org/10.1115/imece2005-82012.
Texte intégralRésumé :
In consultation with ASTM and other stakeholders in Tissue-Engineered Medical Products (TEMPs) industry, the National Institute of Standards and Technology (NIST) initiated a project designed to produce Reference Material scaffolds for tissue engineering. The rationale for Reference Material scaffolds was developed through several NIST/Industry workshops. In brief, Reference Material scaffolds have multiple uses: facilitating the development and the validation of new test methods that measure interactions among various components of a TEMP; comparison with other scaffolds and scaffold materials in terms of cellular responses, biodegradation, and releases of growth factors; and comparisons of responses among various cell lines. The primary customers for Reference Material scaffolds are expected to be the TEMPs industry, academic researchers, regulators, and standards developing organizations. There are many properties of a TEMP that warrant development of multiple Reference Material scaffolds. Currently, NIST is defining a set of Reference Material scaffolds based on geometric descriptors such as permeability, pore volume, pore size distribution, interconnectivity, and tortuosity. In consultation with ASTM, NIST is testing three candidate scaffolds produced by: three dimensional (3-D) printing, stereolithography, and fused deposition modeling (FDM). Scaffolds made by these methods have been obtained from Mayo Clinic (Rochester, MN), Case Western Reserve University (CWRU) (Cleveland, OH), and Osteopore International (Singapore), respectively, for structural characterization. These prototype scaffolds, with well-defined architectures, have been selected to address the following items of interest: 1) establishment of useful functional definitions of porosity content, interconnectivity, and pores; 2) evaluation of testing methods listed in the Standard Guide for the Porosity of Polymeric Scaffolds for Use in Tissue-Engineered Medical Products, which is being drafted by ASTM. Currently, NIST and the Center for Devices and Radiological Health of the Food and Drug Administration, as well as other groups from US and foreign laboratories, are actively carrying out cross-validation test of these prototype scaffolds.
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Ay, Emrah, et Nizami Duran. « Resistance of SARS CoV-2 to Seawater ». Dans The 9th International Conference on Advanced Materials and Systems. INCDTP - Leather and Footwear Research Institute (ICPI), Bucharest, Romania, 2022. http://dx.doi.org/10.24264/icams-2022.iii.2.
Texte intégralRésumé :
SARS CoV-2, which is the cause of Covid-19 disease, has become the only and most important agenda of the world due to its mortality and morbidity that globally affects the whole world. The virus has profoundly affected life all over the world. The lifestyles of people have changed due to the virus. This study is planned to understand how important sea water is in SARS-CoV-2 transmission. The study aimed to determine whether there is a risk of sea water in SARS-CoV-2 transmission. The effectiveness of seawater on SARS CoV-2 viability has been investigated in different dilutions of seawater in different time periods. Experiments were carried out in three different titrations of SARS CoV-2 in Vero cell lines. Viral replication has been investigated by detecting morphological changes occurring in cells, cell viability, and the RT-PCR method. Seawater has been found to be highly potent inhibitory on SARS CoV-2 about time and dose. Especially within 300 seconds, seawater has been found to inhibit viral replication up to 1/32 dilution. These results show that viral transmission through seawater is quite difficult for people swimming in the sea during the pandemic. Seawater-mediated spread of SARS-CoV-2 is out of the question. However, these results should not be interpreted as the prophylactic activity of saline against viruses, which are obligate intracellular parasites.
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Bonotto, Nathália Cardoso De Afonso, Bárbara Osmarin Turra, Cibele Ferreira Teixeira, Ivana Beatrice Mânica Cruz et Fernanda Barbisan. « QUETIAPINA NÃO-METABOLIZADA MODULA A FORMAÇÃO DE ARMADILHAS EXTRACELULARES DE NEUTRÓFILOS ». Dans I Congresso Brasileiro de Imunologia On-line. Revista Multidisciplinar em Saúde, 2021. http://dx.doi.org/10.51161/rems/997.
Texte intégralRésumé :
Introdução: A quetiapina (QUE) é um fármaco que, devido as suas diferentes formas de interação com os receptores, apresenta grande aplicabilidade clínica. Entretanto, esta droga possui muitos efeitos indesejados, tais como distúrbios da via metabólica e alterações no sistema imunológico. Durante seu metabolismo, 95% da QUE é biotransformada em seu principal metabólito ativo, a norquetiapina, os outros 5% permanecem como quetiapina não metabolizada (nmQUE), esta que pode ser responsável por alterações imunológicas periféricas, principalmente a inflamação crônica de baixo grau. Os neutrófilos são a principal linha de defesa dentre os glóbulos brancos, estando inteiramente associados à resposta imune inata. Recentemente, descobriu-se que essas células são capazes de formar uma estrutura única de DNA “decorada” de peptídeos antimicrobianos, denominadas armadilhas extracelulares de neutrófilos (NETs), as quais se formam logo após a hiperestimulação. A formação das NETs durante as doenças inflamatórias é favorecida por tecidos pró-inflamatórios e citocinas sistêmicas. Objetivo: Avaliar in vitro o possível efeito modulatório da nmQUE na ativação inflamatória causada pela formação de NETs. Métodos: Neutrófilos isolados do sangue humano de doadores saudáveis foram previamente ativados ou não pela exposição a células de leveduras (Saccharomyces cerevisiae), concentração de 1x107 cels/mL por 2 horas. Posteriormente, as células foram expostas a 100 µg/L de nmQUE por mais 2 horas, sendo, logo após, fixadas e coradas via Kit Panótico® e reagente Quant-iT™ PicoGreen®, seguidos pela captura de imagens. A formação das NETs foi mensurada via software Digimizer Image Analysis seguida pela análise não-paramétrica de Kruskal Wallis. Os valores foram considerados significativos quando p≤0,05. Resultados: Nas células tratadas apenas com nmQUE foi possível observar uma maior indução da formação de NETs, quando comparadas aquelas expostas somente à levedura. A ativação das NETs foi ainda maior no grupo exposto tanto a células de leveduras, quanto a nmQUE. Conclusão: Apesar das limitações inerentes aos estudos in vitro, nossos resultados demonstram que a nmQUE apresentou influência significativa na formação de NETs, com e sem a exposição a células de leveduras, representando um grande problema da terapia antipsicótica.
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Pereira, Júlia de Paula, FABIANA MARIA OLIVEIRA BAÊTA, JOÃO VITOR FREITAS LANZA AVELAR, NILCIANY APARECIDA DE SOUSA RIBEIRO et ALVARO FERNANDO SILVA NASCIMENTO. « O PAPEL DAS CÉLULAS NKS NA RESPOSTA IMUNOLÓGICA CONTRA O VÍRUS SARS-COV-2 ». Dans II Congresso Brasileiro de Imunologia On-line. Revista Multidisciplinar em Saúde, 2022. http://dx.doi.org/10.51161/ii-conbrai/6038.
Texte intégralRésumé :
Introdução: A COVID-19, doença causada pelo vírus SARS-CoV-2 tornou-se pandêmica em 11 de março de 2020 (OMS). Até 28 de fevereiro de 2022, foram contabilizadas 5,95 milhões de mortes pela COVID-19. Apesar desse número, várias pessoas se recuperaram da doença antes de serem imunizadas. Isso se deve também à formação de uma resposta imunológica natural ou inata. Dentre os mecanismos da resposta inata, podemos destacar o papel das células NKs, que por desempenharem funções importantes em outras infecções virais, espera-se também sua atividade na COVID-19, visto sua capacidade de eliminação viral em âmbito intracelular. Objetivos: Avaliar a relação das células NKs nas infecções causadas pelo SARS-CoV-2 no âmbito da resposta inata. Metodologia: Realizou-se uma revisão da literatura em trabalhos disponíveis no PubMed, usando o operador booleano “and” para associar os descritores “COVID-19”, “Innate immunity”, “NKs cells”, “respiratory”. Na busca feita no dia 23 de fevereiro de 2022, utilizou-se trabalhos publicados entre os anos de 2020 e 2022. Foram obtidos 54 artigos, sendo selecionados 34 trabalhos de acordo com os seguintes critérios de exclusão: trabalhos que não estavam relacionados, de alguma forma, à COVID-19, à resposta inata e às células NKs. Resultados: Estudos com pacientes em diferentes estágios da COVID-19 demonstraram que alterações imunológicas são frequentes independente da gravidade da doença. Porém, quando restringimos essas alterações às células NK, percebemos que ela está presente principalmente em pacientes com doença severa. A quantidade de células NKs se encontram reduzidas em casos graves e essa persistência está associada a um quadro de fibrose pulmonar. Ademais, pacientes em processo de recuperação da COVID-19 têm aumento na quantidade destas células, assim como de seus mecanismos efetores. Conclusão: As células NKs têm papel central na resposta inicial contra o SARS-CoV-2, eliminando células infectadas e modulando a resposta imunológica subsequente pela liberação de citocinas. Porém, em pacientes com quadros graves, as NKs estão em menor quantidade e menos ativas, o que pode explicar a severidade destes pacientes. Assim, as NKs podem ser utilizadas como marcadores da doença severa e o restabelecimento de sua atividade e proliferação, podem ser utilizados como forma de evitar o agravamento da COVID-19.
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Trinnye, Luizze Santos, Bruna Carolina Rangel Fortes, Caroline Domingues Zwicker et Gabriele Soares Martins. « IMPLICAÇÕES FISIOPATOLÓGICAS DO COVID-19 NA ANEMIA FALCIFORME : UMA REVISÃO BIBLIOGRÁFICA ». Dans I Congresso Brasileiro de Hematologia Clínico-laboratorial On-line. Revista Multidisciplinar em Saúde, 2021. http://dx.doi.org/10.51161/rems/617.
Texte intégralRésumé :
Introdução: A anemia falciforme consiste em um distúrbio hereditário monogênico, caracterizado por lesões endoteliais hemolíticas e crises vaso-oclusivas, sendo a forma mais prevalente dentre as manifestações falciformes. Ao longo da atual pandemia, a associação de tal patologia com a infecção pelo SARS-CoV-2 foi considerada um risco potencial, sobretudo devido à incidência de comprometimento vascular e esplênico. Objetivo: Relatar e comparar as evidências literárias dos impactos fisiopatológicos sistêmicos resultantes da infecção por COVID-19 nos casos de anemia falciforme. Material e métodos: Consiste em uma Revisão de Literatura, sendo encontrados 69 artigos e selecionados 18, correspondentes a Metanálises, Revisões Narrativas e Séries de casos. Buscou-se nas bases de dados PUBMED, LILACS e ScienceDirect. Foram incluídas publicações do ano de 2020, utilizando os descritores “Doença Falciforme” (sickle cell disease), “COVID-19” e “SARS-CoV-2” associados ao operador booleano ‘’AND’’. Artigos com viés e repetidos foram excluídos. Resultados: Observou-se que em função da vulnerabilidade imunológica a qual os pacientes com doença falciforme (DF) estão sujeitos, a COVID-19 pode atuar como gatilho para suscitar complicações potencialmente graves. Em análise, avaliou-se o impacto do vírus em 4 pacientes com anemia falciforme nos EUA, de forma que todos desenvolveram crises de dor aguda, enquanto no Reino Unido, uma série de casos revelou 75% de prevalência para crises vaso-oclusivas (CVO) por infecção viral. Pesquisas brasileiras, por sua vez, demonstraram maior susceptibilidade para os quadros de síndrome torácica aguda (STA) e acometimento discreto para o tromboembolismo pulmonar. Em geral, há uma evolução prognóstica favorável nos quadros de DF isentos de comorbidades, entretanto, são necessárias maiores evidências para comprovação. Conclusão: Os estudos evidenciaram que a infecção por COVID-19 em pacientes com anemia falciforme aumenta o risco de tromboembolismo, STA e CVO. Logo, pacientes com DF e infecção viral por SARS-CoV-2 merecem ser melhor avaliados, necessitando-se, assim, de maiores evidências clínicas elucidativas.
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Sundararajan, Desikan, et Abdul-Majeed Azad. « Development of Logistic Fuel Desulfurizers Endowed With Nanoartifacts ». Dans ASME 2008 6th International Conference on Fuel Cell Science, Engineering and Technology. ASMEDC, 2008. http://dx.doi.org/10.1115/fuelcell2008-65138.
Texte intégralRésumé :
Sulfur content in the logitstic fuels such as diesel, jet fuel and coal poses great challenge, as it leads to severe deactivation and poisioning of the reforming catalysts. The utilization of logistic fuel in high efficiency fuel cells as hydrogen-rich reformates, therefore, necessitates that the sulfur (mostly present as organosulfur species) be either eliminated totally, or its level be reduced below such levels as not to mitigate the long-term sustained performance of the reforming catalysts. Besides, SOFC anodes are also quickly poisoned by sulfur present even in trace amounts (1–2 ppm). Thus, desulfurization becomes an important and integral component in harnessing clean power from these abundunt fuel resources. Recently, we have developed a series of novel sulfur sorbents with agile scavengers dispersed thoroughly and uniformly on lightweight, highly periodic nanoporous biomimetic or zeolitic inert matrices. These formulations have been tested in a range of temperatures for durations ranging from 12 to 100 hours in sulfur-bearing gas streams. In this presentation, their synthesis, characterization (before and after sulfidation) by XRD, SEM/EDS, RBSE imaging, on-line real-time quantification in gas chromatography in conjunction with systematic chemical analyses to gauge their efficacy in sulfur removal will be discussed.
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Zhang, Qingwei, Ioannis Neitzel, Vadym N. Mochalin, Isabel Knoke, David M. Wootton, Yury Gogotsi, Peter I. Lelkes et Jack G. Zhou. « PLLA-Nanodiamond Composites and Their Application in Bone Tissue Engineering ». Dans ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13336.
Texte intégralRésumé :
Nanodiamond (ND) is an attractive nanomaterial for reinforcement of polymers [1] due to the ND’s superior mechanical and chemical properties, and low biotoxicity. A novel composite material has been produced for bone scaffolds utilizing the biodegradable polymer, poly(L-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA) [2]. Composites were prepared by admixing to a PLLA/chloroform solution chloroform suspensions of ND-ODA at concentrations of 0, 1, 3, 5, 7, and 10 (w/w). Dispersion of ND-ODA in the composites was studied by transmission electron microscopy (TEM). The lower-resolution TEM micrograph of 1% wt ND-ODA/PLLA film (Fig. 1a) shows nanodiamond particles dispersed in PLLA film on amorphous carbon support. Due to long hydrocarbon chains of ODA the ND-ODA particles have good wettability with the PLLA so there is no segregation of ND-ODA and PLLA, and the polymer completely surrounds the particles. The high-resolution TEM image (Fig. 1b) shows ND crystals with attached organic material that can be ODA or PLLA. Nanoindentation tests show that the mechanical strength of ND-ODA/PLLA composites improves upon addition of ND (Table 1). Even at low concentrations (1% wt) the ND-ODA increased the hardness of the composite by 60% and Young’s modulus by 20% over neat PLLA. Based on our preliminary observations, we conclude that further additions of ND-ODA resulted in smaller changes with subsequent saturation in the mechanical properties at ∼7% wt (see Table 1). ND is relatively novel nanomaterial. Establishing its biocompatibility requires further studies, especially for modified ND. We studied the biocompatibility of 5–10nm ND and ND-ODA in experiments with a murine osteoblast cell line (7F2, from ATCC). Incubation of a cultured osteoblasts with 1–100μg/ml of ND or ND-ODA particles for 4 hours did not show much influence on the cell viability (Fig. 2), as inferred from an alamarBlue™ assay. To test the feasibility of ND-ODA/PLLA as a matrix material supporting cell growth, osteoblasts were cultured on the composites for 6 days. The attactment and proliferation of 7F2 cells on the scaffolds were assessed, respectively, by fluorescent nuclear staining with Hoechst 33258 and the alamarBlueTM assay. Our results showed that the addition of ND-ODA had only a negligibly small effect on cell proliferation, which is indicative of good biocompatibility of the composites (Fig. 3). The morphology of 7F2 cells growing on all ND-ODA/PLLA composite scaffolds was assessed by SEM. The data (not shown) confirm that the osteoblasts spread on the scaffolds similar to their spreading on TCP (tissue culture plastic). To summarize, the improved mechanical properties of the PLLA/ND-ODA composites and their good biocompatibility suggest that these materials may be suitable for applications in musculoskeletal tissue engineering.
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Sidarta, Djoni E., Nicolas Tcherniguin, Philippe Bouchard et Ho-Joon Lim. « Sensitivity Analysis of an ANN-Based System for Detection of Mooring Line Failure ». Dans ASME 2020 39th International Conference on Ocean, Offshore and Arctic Engineering. American Society of Mechanical Engineers, 2020. http://dx.doi.org/10.1115/omae2020-18818.
Texte intégralRésumé :
Abstract Monitoring the integrity of mooring lines on floating offshore platforms is one of the key factors in ensuring safe and productive offshore operations. Sensors, such as inclinometers, compressive load cells or strain sensors, can be used to monitor the inclination angles or tensions on mooring lines. An alternative method using only dry monitoring systems, such as DGPS (Differential Global Positioning System), Gyrocompass and/or IMU (Inertial Measurement / Motion Unit), can also be used to monitor the integrity of mooring lines. This method uses the measured motions and positions of a vessel without any information on the environmental conditions to detect mooring line failure. The detection of mooring line failure is based on detecting shifts in low-frequency periods and mean yaw angles as a function of vessel position, mass and added mass. The proposed method utilizes Artificial Neural Network (ANN) to recognize and classify patterns. The training of an ANN model requires examples/data associated with intact mooring lines and broken mooring line(s). Examples/data of broken mooring line(s) are practically available only from numerical simulations. Therefore, it is important to address these two key topics: (1) Is the real behavior of the floating offshore platform sufficiently aligned with numerical simulations? and (2) The effect of the accuracy of monitoring equipment on the performance of an ANN-based system. The first topic is reviewed briefly with its possible solution including some sensitivity tests, and this paper focuses on addressing the second topic. A system architecture is discussed in this paper along with the accuracy of the monitoring equipment. As an example, an ANN model has been trained to detect a broken mooring line of a spread-moored FPSO. This ANN model has been tested on its performance in dealing with a range of possible errors associated with the monitoring equipment. Furthermore, the tests have been carried out for a combination of variables that are not included in the ANN training, such as: vessel draft (mass), sea state conditions and directions. This paper presents the results of the tests for various variable sensitivities, which cover vessel positions, mean yaw angles and vessel drafts. These are essentially testing the tolerance of a trained ANN model against error or noise in the data. The results show that a trained ANN model can be error/noise tolerant.
Rapports d'organisations sur le sujet "SET-2 cell lines"
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Ohad, Nir, et Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, janvier 2004. http://dx.doi.org/10.32747/2004.7695869.bard.
Texte intégralRésumé :
Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas et Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, janvier 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
Texte intégralRésumé :
The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Chejanovsky, Nor, et Bruce A. Webb. Potentiation of pest control by insect immunosuppression. United States Department of Agriculture, juillet 2004. http://dx.doi.org/10.32747/2004.7587236.bard.
Texte intégralRésumé :
Our original aims were to elucidate the mechanisms through which the immunosuppressive insect virus, the Campoletis sonorensis polydnavirus (CsV) promotes replication of a well-characterized pathogenic virus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in hosts that are mildly or non-permissive to virus replication. According to the BARD panels criticism we modified our short-term goals (see below). Thus, in this feasibility study (one-year funding) we aimed to show that: 1. S. littoralis larvae mount an immune response against a baculovirus infection. 2. Immunosuppression of an insect pest improves the ability of a viral pathogen (a baculovirus) to infect the pest. 3. S. littoralis cells constitute an efficient tool to study some aspects of the anti- viral immune response. We achieved the above objectives by: 1. Finding melanized viral foci upon following the baculoviral infection in S . littoralis larvae infected with a polyhedra - positive AcMNPV recombinant that expressed the GFP gene under the control of the Drosophila heat shock promoter. 2. Studying the effect of AcMNPV-infection in S . littoralis immunosuppressed by parasitation with the Braconidae wasp Chelonus inanitus that bears the CiV polydna virus, that resulted in higher susceptibility of S. littoralis to AcMNPV- infection. 3. Proving that S. littoralis hemocytes resist AcMNPV -infection. 4. Defining SL2 as a granulocyte-like cell line and demonstrating that as littoralis hemocytic cell line undergoes apoptosis upon AcMNPV -infection. 5. Showing that some of the recombinant AcMNPV expressing the immuno-suppressive polydna virus CsV- vankyrin genes inhibit baculoviral-induced lysis of SL2 cells. This information paves the way to elucidate the mechanisms through which the immuno- suppressive polydna insect viruses promote replication of pathogenic baculoviruses in lepidopteran hosts that are mildly or non-permissive to virus- replication by: - Assessing the extent to which and the mechanisms whereby the immunosuppressive viruses, CiV and CsV or their genes enhance AcMNPV replication in polydnavirus- immunosuppressed H. zea and S. littoralis insects and S. littoralis cells. - Identifying CiV and CsV genes involved in the above immunosuppression (e.g. inhibiting cellular encapsulation and disrupting humoral immunity). This study will provide insight to the molecular mechanisms of viral pathogenesis and improve our understanding of insect immunity. This knowledge is of fundamental importance to controlling insect vectored diseases of humans, animals and plants and essential to developing novel means for pest control (including baculoviruses) that strategically weaken insect defenses to improve pathogen (i.e. biocontrol agent) infection and virulence.
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Blum, Abraham, Henry T. Nguyen et N. Y. Klueva. The Genetics of Heat Shock Proteins in Wheat in Relation to Heat Tolerance and Yield. United States Department of Agriculture, août 1993. http://dx.doi.org/10.32747/1993.7568105.bard.
Texte intégralRésumé :
Fifty six diverse spring wheat cultivars were evaluated for genetic variation and heritability for thermotolerance in terms of cell-membrane stability (CMS) and triphenyl tetrazolium chloride (TTC) reduction. The most divergent cultivars for thermotolerance (Danbata-tolerant and Nacozari-susceptible) were crossed to develop an F8 random onbred line (RIL) population. This population was evaluated for co-segragation in CMS, yield under heat stress and HSP accumulation. Further studies of thermotolerance in relations to HSP and the expression of heterosis for growth under heat stress were performed with F1 hybrids of wheat and their parental cultivars. CMS in 95 RILs ranged from 76.5% to 22.4% with 71.5% and 31.3% in Danbata and Nacozari, respectively. The population segregated with a normal distribution across the full range of the parental values. Yield and biomass under non-stress conditions during the normal winter season at Bet Dagan dit not differ between the two parental cultivar, but the range of segregation for these traits in 138 RILs was very high and distinctly transgressive with a CV of 35.3% and 42.4% among lines for biomass and yield, respectively. Mean biomass and yield of the population was reduced about twofold when grown under the hot summer conditions (irrigated) at Bet Dagan. Segregation for biomass and yield was decreased relative to the normal winter conditions with CV of 20.2% and 23.3% among lines for biomass and yield, respectively. However, contrary to non-stress conditions, the parental cultivars differed about twofold in biomass and yield under heat stress and the population segregated with normal distribution across the full range of this difference. CMS was highly and positively correlated across 79 RILs with biomass (r=0.62**) and yield (r=0.58**) under heat stress. No such correlation was obtained under the normal winter conditions. All RILs expressed a set of HSPs under heat shock (37oC for 2 h). No variation was detected among RILs in high molecular weight HSP isoforms and they were similar to the patterns of the parental cultivars. There was a surprisingly low variability in low molecular weight HSP isoforms. Only one low molecular weight and Nacozari-specific HSP isoform (belonging to HSP 16.9 family) appeared to segregate among all RILs, but it was not quantitatively correlated with any parameter of plant production under heat stress or with CMS in this population. It is concluded that this Danbata/Nacozari F8 RIL population co-segregated well for thermotolerance and yield under heat stress and that CMS could predict the relative productivity of lines under chronic heat stress. Regretfully this population did not express meaningful variability for HSP accumulation under heat shock and therefore no role could be seen for HSP in the heat tolerance of this population. In the study of seven F1 hybrids and their parent cultivars it was found that heterosis (superiority of the F1 over the best parent) for CMs was generally lower than that for growth under heat stress. Hybrids varied in the rate of heterosis for growth at normal (15o/25o) and at high (25o/35o) temperatures. In certain hybrids heterosis for growth significantly increased at high temperature as compared with normal temperature, suggesting temperature-dependent heterosis. Generally, under normal temperature, only limited qualitative variation was detected in the patterns of protein synthesis in four wheat hybrids and their parents. However, a singular protein (C47/5.88) was specifically expressed only in the most heterotic hybrid at normal temperature but not in its parent cultivars. Parental cultivars were significantly different in the sets of synthesized HSP at 37o. No qualitative changes in the patterns of protein expression under heat stress were correlated with heterosis. However, a quantitative increase in certain low molecular weight HSP (mainly H14/5.5 and H14.5.6, belonging to the HSP16.9 family) was positively associated with greater heterosis for growth at high temperature. None of these proteins were correlated with CMS across hybrids. These results support the concept of temperature-dependent heterosis for growth and a possible role for HSP 16.9 family in this respect. Finally, when all experiments are viewed together, it is encouraging to find that genetic variation in wheat yield under chronic heat stress is associated with and well predicted by CMS as an assay of thermotolerance. On the other hand the results for HSP are elusive. While very low genetic variation was expressed for HSP in the RIL population, a unique low molecular weight HSP (of the HSP 16.9 family) could be associated with temperature dependant heterosis for growth.
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Sessa, Guido, et Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, janvier 2004. http://dx.doi.org/10.32747/2004.7695876.bard.
Texte intégralRésumé :
The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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Funkenstein, Bruria, et Shaojun (Jim) Du. Interactions Between the GH-IGF axis and Myostatin in Regulating Muscle Growth in Sparus aurata. United States Department of Agriculture, mars 2009. http://dx.doi.org/10.32747/2009.7696530.bard.
Texte intégralRésumé :
Growth rate of cultured fish from hatching to commercial size is a major factor in the success of aquaculture. The normal stimulus for muscle growth in growing fish is not well understood and understanding the regulation of muscle growth in fish is of particular importance for aquaculture. Fish meat constitutes mostly of skeletal muscles and provides high value proteins in most people's diet. Unlike mammals, fish continue to grow throughout their lives, although the size fish attain, as adults, is species specific. Evidence indicates that muscle growth is regulated positively and negatively by a variety of growth and transcription factors that control both muscle cell proliferation and differentiation. In particular, growth hormone (GH), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs) and transforming growth factor-13 (TGF-13) play critical roles in myogenesis during animal growth. An important advance in our understanding of muscle growth was provided by the recent discovery of the crucial functions of myostatin (MSTN) in controlling muscle growth. MSTN is a member of the TGF-13 superfamily and functions as a negative regulator of skeletal muscle growth in mammals. Studies in mammals also provided evidence for possible interactions between GH, IGFs, MSTN and the musclespecific transcription factor My oD with regards to muscle development and growth. The goal of our project was to try to clarify the role of MSTNs in Sparus aurata muscle growth and in particular determine the possible interaction between the GH-IGFaxis and MSTN in regulating muscle growth in fish. The steps to achieve this goal included: i) Determining possible relationship between changes in the expression of growth-related genes, MSTN and MyoD in muscle from slow and fast growing sea bream progeny of full-sib families and that of growth rate; ii) Testing the possible effect of over-expressing GH, IGF-I and IGF-Il on the expression of MSTN and MyoD in skeletal muscle both in vivo and in vitro; iii) Studying the regulation of the two S. aurata MSTN promoters and investigating the possible role of MyoD in this regulation. The major findings of our research can be summarized as follows: 1) Two MSTN promoters (saMSTN-1 and saMSTN-2) were isolated and characterized from S. aurata and were found to direct reporter gene activity in A204 cells. Studies were initiated to decipher the regulation of fish MSTN expression in vitro using the cloned promoters; 2) The gene coding for saMSTN-2 was cloned. Both the promoter and the first intron were found to be polymorphic. The first intron zygosity appears to be associated with growth rate; 3) Full length cDNA coding for S. aurata growth differentiation factor-l I (GDF-II), a closely related growth factor to MSTN, was cloned from S. aurata brain, and the mature peptide (C-terminal) was found to be highly conserved throughout evolution. GDF-II transcript was detected by RT -PCR analysis throughout development in S. aurata embryos and larvae, suggesting that this mRNA is the product of the embryonic genome. Transcripts for GDF-Il were detected by RT-PCR in brain, eye and spleen with highest level found in brain; 4) A novel member of the TGF-Bsuperfamily was partially cloned from S. aurata. It is highly homologous to an unidentified protein (TGF-B-like) from Tetraodon nigroviridisand is expressed in various tissues, including muscle; 5) Recombinant S. aurata GH was produced in bacteria, refolded and purified and was used in in vitro and in vivo experiments. Generally, the results of gene expression in response to GH administration in vivo depended on the nutritional state (starvation or feeding) and the time at which the fish were sacrificed after GH administration. In vitro, recombinantsaGH activated signal transduction in two fish cell lines: RTHI49 and SAFI; 6) A fibroblastic-like cell line from S. aurata (SAF-I) was characterized for its gene expression and was found to be a suitable experimental system for studies on GH-IGF and MSTN interactions; 7) The gene of the muscle-specific transcription factor Myogenin was cloned from S. aurata, its expression and promoter activity were characterized; 8) Three genes important to myofibrillogenesis were cloned from zebrafish: SmyDl, Hsp90al and skNAC. Our data suggests the existence of an interaction between the GH-IGFaxis and MSTN. This project yielded a great number of experimental tools, both DNA constructs and in vitro systems that will enable further studies on the regulation of MSTN expression and on the interactions between members of the GHIGFaxis and MSTN in regulating muscle growth in S. aurata.
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Horwitz, Benjamin, et Nicole M. Donofrio. Identifying unique and overlapping roles of reactive oxygen species in rice blast and Southern corn leaf blight. United States Department of Agriculture, janvier 2017. http://dx.doi.org/10.32747/2017.7604290.bard.
Texte intégralRésumé :
Plants and their fungal pathogens both produce reactive oxygen species (ROS). CytotoxicROS act both as stressors and signals in the plant-fungal interaction. In biotrophs, a compatible interaction generates little ROS, but is followed by disease. An incompatible interaction results in a strong oxidative burst by the host, limiting infection. Necrotrophs, in contrast, thrive on dead and dying cells in an oxidant-rich local environment. Rice blast, Magnaportheoryzae, a hemibiotroph, occurs worldwide on rice and related hosts and can decimate enough rice each year to feed sixty million people. Cochliobolusheterostrophus, a necrotroph, causes Southern corn leaf blight (SLB), responsible for a major epidemic in the 1970s. The objectives of our study of ROS signaling and response in these two cereal pathogens were: Confocal imaging of ROS production using genetically encoded redox sensor in two pathosystems over time. Forward genetic screening of HyPer sensor lines in two pathosystems for fungal genes involved in altered ROSphenotypes. RNA-seq for discovery of genes involved in ROS-related stress and signaling in two pathosystems. Revisions to the research plan: Library construction in SLB was limited by low transformation efficiency, compounded by a protoplasting enzyme being unavailable during most of year 3. Thus Objective 2 for SLB re-focused to construction of sensor lines carrying deletion mutations in known or candidate genes involved in ROS response. Imaging on rice proved extremely challenging, so mutant screening and imaging were done with a barley-infecting line, already from the first year. In this project, ROS imaging at unprecedented time and spatial resolution was achieved, using genetically-encoded ratio sensors in both pathogens. This technology is currently in use for a large library of rice blast mutants in the ROS sensor background, and Southern corn leaf blight mutants in final stages of construction. The imaging methods developed here to follow the redox state of plant pathogens in the host tissue should be applicable to fungal pathogens in general. Upon completion of mutant construction for SCLB we hope to achieve our goal of comparison between intracellular ROS status and response in hemibiotroph and necrotroph cereal pathogens.
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Fluhr, Robert, et Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, janvier 2012. http://dx.doi.org/10.32747/2012.7697123.bard.
Texte intégralRésumé :
Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Sherman, Amir, Rebecca Grumet, Ron Ophir, Nurit Katzir et Yiqun Weng. Whole genome approach for genetic analysis in cucumber : Fruit size as a test case. United States Department of Agriculture, décembre 2013. http://dx.doi.org/10.32747/2013.7594399.bard.
Texte intégralRésumé :
The Cucurbitaceae family includes a broad array of economically and nutritionally important crop species that are consumed as vegetables, staple starches and desserts. Fruit of these species, and types within species, exhibit extensive diversity as evidenced by variation in size, shape, color, flavor, and others. Fruit size and shape are critical quality determinants that delineate uses and market classes and are key traits under selection in breeding programs. However, the underlying genetic bases for variation in fruit size remain to be determined. A few species the Cucurbitaceae family were sequenced during the time of this project (cucumber was already sequenced when the project started watermelon and melon sequence became available during the project) but functional genomic tools are still missing. This research program had three major goals: 1. Develop whole genome cucumber and melon SNP arrays. 2. Develop and characterize cucumber populations segregating for fruit size. 3. Combine genomic tools, segregating populations, and phenotypic characterization to identify loci associated with fruit size. As suggested by the reviewers the work concentrated mostly in cucumber and not both in cucumber and melon. In order to develop a SNP (single nucleotide polymorphism) array for cucumber, available and newly generated sequence from two cucumber cultivars with extreme differences in shape and size, pickling GY14 and Chinese long 9930, were analyzed for variation (SNPs). A large set of high quality SNPs was discovered between the two parents of the RILs population (GY14 and 9930) and used to design a custom SNP array with 35000 SNPs using Agilent technology. The array was validated using 9930, Gy14 and F1 progeny of the two parents. Several mapping populations were developed for linkage mapping of quantitative trait loci (QTL) for fruit size These includes 145 F3 families and 150 recombinant inbred line (RILs F7 or F8 (Gy14 X 9930) and third population contained 450 F2 plants from a cross between Gy14 and a wild plant from India. The main population that was used in this study is the RILs population of Gy14 X 9930. Phenotypic and morphological analyses of 9930, Gy14, and their segregating F2 and RIL progeny indicated that several, likely independent, factors influence cucumber fruit size and shape, including factors that act both pre-anthesis and post-pollination. These include: amount, rate, duration, and plane of cell division pre- and post-anthesis and orientation of cell expansion. Analysis of F2 and RIL progeny indicated that factors influencing fruit length were largely determined pre-anthesis, while fruit diameter was more strongly influenced by environment and growth factors post-anthesis. These results suggest involvement of multiple genetically segregating factors expected to map independently onto the cucumber genome. Using the SNP array and the phenotypic data two major QTLs for fruit size of cucumber were mapped in very high accuracy (around 300 Kb) with large set of markers that should facilitate identification and cloning of major genes that contribute to fruit size in cucumber. In addition, a highly accurate haplotype map of all RILS was created to allow fine mapping of other traits segregating in this population. A detailed cucumber genetic map with 6000 markers was also established (currently the most detailed genetic map of cucumber). The integration of genetics physiology and genomic approaches in this project yielded new major infrastructure tools that can be used for understanding fruit size and many other traits of importance in cucumber. The SNP array and genetic population with an ultra-fine map can be used for future breeding efforts, high resolution mapping and cloning of traits of interest that segregate in this population. The genetic map that was developed can be used for other breeding efforts in other populations. The study of fruit development that was done during this project will be important in dissecting function of genes that that contribute to the fruit size QTLs. The SNP array can be used as tool for mapping different traits in cucumber. The development of the tools and knowledge will thus promote genetic improvement of cucumber and related cucurbits.
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Funkenstein, Bruria, et Cunming Duan. GH-IGF Axis in Sparus aurata : Possible Applications to Genetic Selection. United States Department of Agriculture, novembre 2000. http://dx.doi.org/10.32747/2000.7580665.bard.
Texte intégralRésumé :
Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.