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1
Garriga, Carlos, Aaron Hedlund, Yang Tang und Ping Wang. „Rural-Urban Migration, Structural Transformation, and Housing Markets in China“. American Economic Journal: Macroeconomics 15, Nr. 2 (01.04.2023): 413–40. http://dx.doi.org/10.1257/mac.20160142.
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This paper investigates the interrelationship between urbanization, structural transformation, and the post-2000 Chinese housing boom through the lens of a dynamic spatial equilibrium model that features migration and a rich housing market structure with mortgages. Urbanization and structural transformation emerge as key drivers of China's house price boom, while at the same time rising house prices impede these forces of economic transition. Policies to boost urbanization can be undone by the endogenous price response. Land supply expansion ameliorates this negative feedback. Overall, housing markets powerfully shape the path of economic development. (JEL E23, O18, P23, P25, R23, R31, R58)
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Zhang, Jie Lin, Clyde S. Crumpacker und David T. Scadden. „Primitive Hematopoietic Cells Resist HIV-1 Infection Via P21Waf1/Cip1/Sdi1.“ Blood 108, Nr. 11 (16.11.2006): 490. http://dx.doi.org/10.1182/blood.v108.11.490.490.
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Abstract Hematopoietic stem cells are resistant to HIV-1 infection. We have identified a novel mechanism by which the cyclin-dependent kinase inhibitor, p21Waf1/Cip1/Sdi1 (p21), known for its regulation of stem cell pool size (1,2), restricts HIV-1 infection of primitive hematopoietic cells in a non-cell cycle dependent manner. Knocking down p21 by siRNA increased HIV-1 infection and induction of p21 expression by phorbol ester (TPA) blocked HIV-1 replication. P21 did not affect the overall levels of cDNA synthesis, but significantly blocked viral integration and resulted in marked increase in 2-LTR circles, a surrogate marker of abortive integration. Consistent with these observations, p21 coimmunoprecipitated with viral integrase and both were detected in the preintegration complex (PIC). Furthermore, silencing p27Kip1 and p18INK4C, cyclin dependent kinase inhibitors related to p21 that affect cell cycle, revealed no impact on viral DNA integration. A closely related dual-tropic lentivirus with a distinct integrase, SIVmac-251 and the other cell-intrinsic inhibitors of HIV-1, Trim5a, PML, Murr1, and IFN-a were unaffected by p21. These results indicate a new function for p21, participating in prevention of HIV integration into the cellular genome. Therefore p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain the basis for these cells being an uninfected ‘sanctuary’ in HIV disease.
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Kurhan, Faruk, Gulsum Zuhal Kamis, Hamit Hakan Alp, Emine Fusun Akyuz Cim und Abdullah Atli. „A Cross-Sectional Measurement of Endogenous Oxidative Stress Marker Levels in Obsessive Compulsive Disorder“. Psychiatry and Clinical Psychopharmacology 32, Nr. 3 (03.10.2022): 215–21. http://dx.doi.org/10.5152/pcp.2022.21318.
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Funato, Noriko, Kiyoshi Ohtani, Kimie Ohyama, Takayuki Kuroda und Masataka Nakamura. „Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors“. Molecular and Cellular Biology 21, Nr. 21 (01.11.2001): 7416–28. http://dx.doi.org/10.1128/mcb.21.21.7416-7428.2001.
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ABSTRACT Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWISTand FGFR3 genes.
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Morgan, E. L., M. V. Hobbs und W. O. Weigle. „Lymphocyte activation by the Fc region of immunoglobulin. I. Role of prostaglandins in the down regulation of Fc fragment-induced polyclonal antibody production.“ Journal of Immunology 134, Nr. 4 (01.04.1985): 2247–53. http://dx.doi.org/10.4049/jimmunol.134.4.2247.
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Abstract Fc fragment-, subfragment-, and p23-induced polyclonal antibody production are regulated by endogenous and exogenous PGE. Addition of the PG synthetase inhibitor indomethacin (IM) to murine spleen cell cultures resulted in a significant increase in the amount of Ig secreted. Moreover, addition of exogenous PGE to culture resulted in a marked suppression of IgM and IgG secretion. Splenic adherent macrophages and P388D1 cells release PGE upon stimulation with Fc fragments, subfragments, and p23. The inclusion of IM or aspirin in culture was found to abrogate the ability of Fc fragments to induce PGE release from adherent cells. These results suggest a role for PG in immune complex mediated regulation of immune responses.
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Bhaskar, V. „Sex Selection and Gender Balance“. American Economic Journal: Microeconomics 3, Nr. 1 (01.02.2011): 214–44. http://dx.doi.org/10.1257/mic.3.1.214.
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We model the equilibrium sex ratio when parents can choose the sex of their child. With intrinsic son preference, sex selection results in a male-biased sex ratio. This is inefficient due to a marriage market congestion externality. Medical innovations that facilitate selection aggravate the inefficiency. If son preference arises endogenously, due to population growth causing an excess supply of women on the marriage market, selection may improve welfare. Empirically, sex selection causes an excess of males and reduces welfare in China. In most parts of India, cohort sizes are growing, implying an excess supply of women. (JEL J12, J13, J16, O15, P23)
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Chen, Yi-Cheng, Pin-Yu Kuo, Yu-Chi Chou, Hao-Earn Chong, Yu-Tung Hsieh, Mei-Lin Yang, Chao-Liang Wu, Ai-Li Shiau und Chrong-Reen Wang. „Up-Regulated Expression of Pro-Apoptotic Long Noncoding RNA lincRNA-p21 with Enhanced Cell Apoptosis in Lupus Nephritis“. International Journal of Molecular Sciences 22, Nr. 1 (30.12.2020): 301. http://dx.doi.org/10.3390/ijms22010301.
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Accelerated cell apoptosis with dysregulated long noncoding RNAs is the crucial pathogenesis in lupus nephritis (LN). Pro-apoptotic lincRNA-p21 was studied in LN patients, cell lines with lentivirus-mediated overexpression and CRISPR interference (CRISPRi)-conducted repression, and a mouse model. Clinical samples were from patients and age/sex-matched controls. Expression of lincRNA-p21 and endogenous RNA target miR-181a, were examined in mononuclear and urine cells. Guide RNA sequences targeting lincRNA-p21 were cloned into CRISPRi with dCas9/ Krüppel-associated box (KRAB) domain. LincRNA-p21-silened transfectants were investigated for apoptosis and miR-181a expression. LincRNA-p21-overexpressed cells were evaluated for apoptosis and p53-related down-stream molecules. Balb/C mice were injected with pristane to induce LN and examined for apoptosis and lincRNA-p21. Higher lincRNA-p21 levels were found in LN mononuclear and urine cells, positively correlated with activity. There were lower miR-181a levels in LN mononuclear cells, negatively correlated with activity. Doxorubicin-induced apoptotic cells had up-regulated lincRNA-p21 levels. CRISPRi with dCas9/KARA domain showed efficient repression ability on transcription initiation/elongation. CRISPRi-conducted lincRNA-p21-silenced transfectants displayed reduced apoptosis with up-regulated miR-181a levels, whereas lentivirus-mediated lincRNA-p21-overexpressed cells revealed enhanced apoptosis with up-regulated downstream PUMA/Bax expression. LN mice had glomerular apoptosis with progressive increased lincRNA-p21 levels. Our results demonstrate up-regulated lincRNA-p21 expression in LN, implicating a potential diagnostic marker and therapeutic target.
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Luque, Raul M., Beatriz S. Soares, Xiao-ding Peng, Sonia Krishnan, Jose Cordoba-Chacon, Lawrence A. Frohman und Rhonda D. Kineman. „Use of the Metallothionein Promoter-Human Growth Hormone-Releasing Hormone (GHRH) Mouse to Identify Regulatory Pathways that Suppress Pituitary Somatotrope Hyperplasia and Adenoma Formation due to GHRH-Receptor Hyperactivation“. Endocrinology 150, Nr. 7 (02.04.2009): 3177–85. http://dx.doi.org/10.1210/en.2008-1482.
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Hyperactivation of the GHRH receptor or downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, in which adenomas form relatively late in life, with less than 100% penetrance. Hyperplastic and adenomatous pituitaries of metallothionein promoter-human GHRH transgenic (Tg) mice (4 and > 10 months, respectively) were used to identify mechanisms that may prevent or delay adenoma formation in the presence of excess GHRH. In hyperplastic pituitaries, expression of the late G1/G2 marker Ki67 increased, whereas the proportion of 5-bromo-2′-deoxyuridine-labeled cells (S phase marker) did not differ from age-matched controls. These results indicate cell cycle progression is blocked, with further evidence suggesting that enhanced p27 activity may contribute to this process. For adenomas, formation was associated with loss of p27 activity (nuclear localization and mRNA). Increased endogenous somatostatin (SST) tone may also slow the conversion from hyperplastic to adenomatous state because mRNA levels for SST receptors, sst2 and sst5, were elevated in hyperplastic pituitaries, whereas adenomas were associated with a decline in sst1 and sst5 mRNA. Also, SST-knockout Tg pituitaries were larger and adenomas formed earlier compared with those of SST-intact Tg mice. Unexpectedly, these changes were independent of changes in proliferation rate within the hyperplastic tissue, suggesting that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by quantitative real-time RT-PCR.
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Lisetska, I. S., und M. M. Rozhko. „Determination of endogenous intoxication in teenagers and young adults who smoke“. UKRAINIAN JOURNAL OF PERINATOLOGY AND PEDIATRICS, Nr. 2(90) (30.06.2022): 39–43. http://dx.doi.org/10.15574/pp.2022.90.39.
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The harmful habit of smoking is an urgent and important socio-medical problem that has become an epidemic, including in Ukraine. It is a matter of concern that more than 500,000 young people join this bad habit every year. Today, smoking is a modified risk factor for the formation and progression of many diseases, including dental pathology among different age groups, especially among teenagers and young adults. The oral cavity is the first barrier to tobacco smoke with toxins and carcinogens that are part of it. It is known that the pathogenesis of many diseases is accompanied by a nonspecific generalized response of the organism in the form of endogenous intoxication (EI) syndrome, the severity of which may be a criterion for the severity of the pathological process and affect its course. Medium-weight molecules (MWM) are a common marker of EI in biological fluids among metabolites that can be used to assess the severity of disease. The universally accepted marker of EI in biological liquids among metabolites, which gives a possibility to assess the severity of the disease, is medium-weight molecules (MWM) - a class, which combines chemically differently structured components with a mass between 500 and 5000 Da and pronounced biologic activity. Purpose - to determine the degree of EI in the oral fluid by the level of MWM in teenagers and young adults who smoke. Materials and methods. It is studied the dental status of 114 teenagers and young adults aged 15 to 24 years, which was divided into groups: group I included 26 people who regularly smoke traditional cigarettes; group II - 22 people who regularly smoke electronic cigarettes (Vapes); group III - 23 people who regularly smoke tobacco heating devices (IQOS); group IV - 43 people without a bad habit of smoking. The degree of EI was determined by the rate of MWM in oral fluid by the express method according to a modified method Gabrielyan NI et al., 1984. Results. The analyze of the rate in the oral fluid of peptide residues (MWM254) in persons of the group I was exhibited 1.4 times more than in persons of the group IV (p<0.001). There was a similar tendency in the other groups - the group I and the group II had 1.3 times more MWM254 (p<0.05) and 1.2 times more (p<0.001). There was also a difference in the nucleotide fillings (MWM280) in oral fluid of the study groups. Thus, in persons of the group I it was found MWM280 1.6 times more than in persons of the group IV (p<0.001) and 1.3 times more (p<0.05) in persons of the groups II and III respectively. The increase of nucleotide-peptide index was determined depending on the presence and type of malodorous behavior in the study participants. Conclusions. The obtained results indicate the development of EI in teenagers and young adults who smoke, as indicated by the increase in the level of MWM in the oral fluid in the subjects, a marker of endotoxicosis. It was found that the degree of endogenous intoxication depends on the type of smoking, as well as the degree of development of the pathological process. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Keywords: teenagers and young adults, endogenous intoxication, medium weight molecules, smoking.
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Dharmawardhane, Suranganie, Luraynne C. Sanders, Stuart S. Martin, R. Hugh Daniels und Gary M. Bokoch. „Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells“. Journal of Cell Biology 138, Nr. 6 (22.09.1997): 1265–78. http://dx.doi.org/10.1083/jcb.138.6.1265.
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The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics. We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src–transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
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Burdick, Ryan, Jessica L. Smith, Chawaree Chaipan, Yeshitila Friew, Jianbo Chen, Narasimhan J. Venkatachari, Krista A. Delviks-Frankenberry, Wei-Shau Hu und Vinay K. Pathak. „P Body-Associated Protein Mov10 Inhibits HIV-1 Replication at Multiple Stages“. Journal of Virology 84, Nr. 19 (28.07.2010): 10241–53. http://dx.doi.org/10.1128/jvi.00585-10.
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ABSTRACT Recent studies have shown that APOBEC3G (A3G), a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is localized to cytoplasmic mRNA-processing bodies (P bodies). However, the functional relevance of A3G colocalization with P body marker proteins has not been established. To explore the relationship between HIV-1, A3G, and P bodies, we analyzed the effects of overexpression of P body marker proteins Mov10, DCP1a, and DCP2 on HIV-1 replication. Our results show that overexpression of Mov10, a putative RNA helicase that was previously reported to belong to the DExD superfamily and was recently reported to belong to the Upf1-like group of helicases, but not the decapping enzymes DCP1a and DCP2, leads to potent inhibition of HIV-1 replication at multiple stages. Mov10 overexpression in the virus producer cells resulted in reductions in the steady-state levels of the HIV-1 Gag protein and virus production; Mov10 was efficiently incorporated into virions and reduced virus infectivity, in part by inhibiting reverse transcription. In addition, A3G and Mov10 overexpression reduced proteolytic processing of HIV-1 Gag. The inhibitory effects of A3G and Mov10 were additive, implying a lack of functional interaction between the two inhibitors. Small interfering RNA (siRNA)-mediated knockdown of endogenous Mov10 by 80% resulted in a 2-fold reduction in virus production but no discernible impact on the infectivity of the viruses after normalization for the p24 input, suggesting that endogenous Mov10 was not required for viral infectivity. Overall, these results show that Mov10 can potently inhibit HIV-1 replication at multiple stages.
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Dhyani, Anamika, Patricia Favaro und Sara T. Olalla Saad. „ANKHD1 Is Essential for Repair of DNA Double-Strand Breaks in Multiple Myeloma“. Blood 126, Nr. 23 (03.12.2015): 1762. http://dx.doi.org/10.1182/blood.v126.23.1762.1762.
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Abstract ANKHD1, Ankyrin repeat and KH domain-containing protein is highly expressed and plays an important role in the proliferation and cell cycle progression of multiple myeloma (MM) cells. Inhibition of ANKHD1 expression upregulates p21 (CDKN1A, Cyclin Dependent Kinase Inhibitor), a potent cell cycle regulator, and its expression represses p21 promoter. Upregulation of p21 was found to be irrespective of the TP53 mutational status of MM cell lines. A study by our group has shown that ANKHD1 is highly expressed in S phase and that the inhibition of ANKHD1 expression downregulates replication dependent histones suggesting that it might be required for histone transcription (1). Assuming that ANKHD1 might be involved in the transcripitional activation of histones, we studied the effect of ANKHD1 silencing on nuclear protein of the ataxia telangiectasia mutated locus (NPAT), a component of the cell-cycle-dependent histone gene transcription machinery. NPAT associates with histone gene promoters in S phase and suppression of NPAT expression impedes expression of all histone subtypes. In present study, there was a decreased expression of NPAT in ANKHD1 silenced MM cells. Despite the fact that both ANKHD1 and NPAT were localized in the nucleus of MM cells, they did not appear to associate, as observed by confocal microscopy, suggesting at present that ANKHD1 does not modulate histones via NPAT. Since DNA replication is coupled with histone synthesis and downregulation of histones is associated with replication stress and DNA damage, we checked for expression of PCNA (Proliferating Cell Nuclear Antigen), protein involved in DNA replication and repair. PCNA expression was found to be significantly decreased in ANKHD1 inhibited MM cells, suggesting its role in PCNA mediated DNA replication and repair (Fig. 1). To confirm this, we studied the effect of ANKHD1 silencing on some of the components of DNA damage repair (DDR) pathway. We observed increased expression of gamma- H2AX (γ-H2AX i.e Phosphorylated Histone H2AX), marker for DNA double-strand breaks (DSBs) and an early sign of DNA damage induced by replication stress (Fig. 1). We also observed a decrease in phosphorylated CHK2 (Check Point Kinase 2), an essential serine threonine kinase involved in DDR. Accumulation of γ-H2AX on ANKHD1 silencing confirms DNA damage and suggests the possible mechanism of ANKHD1 mediated histones downregulation. In summary, ANKHD1 silencing in MM cells leads to DNA damage (DSBs), suggesting that ANKHD1 is essential for DNA replication and repair. Furthermore, as ANKHD1 negatively regulates p21, and p21 controls DNA replication and repair by interacting with PCNA, we hypothesize that ANKHD1 might be playing role in DNA repair via modulation of the p21-PCNA pathway. Results of the role of ANKHD1 in DNA repair are however preliminary and need to be explored. References: 1) ANKHD1 Is Required for S Phase Progression and Histone Gene Transcription in Multiple Myeloma. Dhyani et al. ASH Abstract; Blood 2014. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Figure 1. Western blot analysis of proteins: a) PCNA and b) γ-H2AX, in control and ANKHD1 silenced U266 MM cell line. Tubulin and GAPDH were used as endogenous controls. Disclosures No relevant conflicts of interest to declare.
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Jane, Stephen, Quan Zhao, Gerhard Rank, Loretta Cerruti, David J. Curtis und John M. Cunningham. „The Protein Methyltransferase Prmt5 Links Histone H4 Methylation to Dnmt3a-Dependent DNA Methylation and Transcriptional Silencing of the Fetal Globin Genes.“ Blood 108, Nr. 11 (16.11.2006): 361. http://dx.doi.org/10.1182/blood.v108.11.361.361.
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Abstract Elevated levels of fetal hemoglobin ameliorate the severity of sickle cell disease and β-thalassemia, fuelling interest in the mechanisms underpinning the fetal (γ) to adult (β) switch in β-like globin chain subtype. We have previously identified a tripartite protein complex consisting of p22 NF-E4, CP2 and ALY, collectively known as the stage selector protein (SSP) that binds to the proximal γ-promoters, and fosters the preferential expression of the γ-genes in fetal erythroid cells. We have also identified a 14 kDa isoform of the NF-E4 protein that plays a role in γ-gene repression by binding CP2 and sequestering it away from the γ-promoter, resulting in disassembly of the activator SSP complex. Despite the loss of SSP binding, we showed by chromatin immunoprecipitation (ChIP) analysis that p22 NF-E4 remained bound to the γ-promoter in this context. To determine whether p22 NF-E4 could serve as the cornerstone for assembly of a larger repressor complex in this setting, we analyzed the proteins that were co-immunoprecipitated with p22 NF-E4 from K562 cell extract by mass spectrometry. One protein identified was PRMT5, an arginine methyltransferase that has been linked to gene silencing by establishing repressive arginine methyl marks including symmetrical dimethylation of arginine 3 on histone H4 (H4R3me2s). We confirmed the interaction between the two endogenous proteins by direct co-immunoprecipitation, and co-localized p22 NF-E4 and PRMT5 to the γ-globin gene promoters by ChIP. In vitro methylation studies using PRMT5 co-immunoprecipitated with p22 NF-E4 confirmed that histone H4 was the major substrate of the enzyme complex in K562 cells. In accord with this, we demonstrated a marked increase in H4R3me2s at the γ-promoter by ChIP in the setting of enforced expression of wild type PRMT5, accompanied by silencing of γ-gene expression. To determine whether additional factors cooperated with PRMT5 in γ-gene repression, we interrogated PRMT5 containing immunoprecipitates with antisera to a range of candidate proteins. We isolated a large repressor complex containing members of the NuRD complex and the methyl domain-binding proteins (MBD2 and MDB3). We also isolated the DNA methyltransferase 3a (Dnmt3a), a finding of considerable interest in view of the links between γ-gene silencing and methylation of CpG dinucleotides. Using bisulfite DNA sequencing, we demonstrated in K562 cells in which PRMT5 expression had been enforced, an increase in the density of methylated CpG dinucleotides clustered around the transcriptional start site. In contrast, cells transfected with an expression vector stably expressing hairpin short interfering RNAs, which induced a 90% reduction in PRMT5 protein levels, showed complete abrogation of DNA methylation at these CpGs, coincident with a five-fold induction of γ-gene expression. ChIP analysis of the human β-globin locus in βYAC transgenic mice revealed a marked enhancement of H4R3me2s at the γ-promoters in adult erythropoietic cells, and absence of this repressive mark at the γ-promoter in the E12.5 fetal liver. This data establishes a direct link between the PRMT5-induced repressive histone mark H4R3me2s and DNA methylation in developmental regulation of γ-gene expression. It also provides impetus for new strategies aimed at reactivation of fetal globin gene expression.
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Poudel, Arati N., Rebekah E. Holtsclaw, Athen Kimberlin, Sidharth Sen, Shuai Zeng, Trupti Joshi, Zhentian Lei et al. „12-Hydroxy-Jasmonoyl-l-Isoleucine Is an Active Jasmonate That Signals through CORONATINE INSENSITIVE 1 and Contributes to the Wound Response in Arabidopsis“. Plant and Cell Physiology 60, Nr. 10 (31.05.2019): 2152–66. http://dx.doi.org/10.1093/pcp/pcz109.
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Abstract 12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.
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Cocoradă, Elena. „Anxiety with and Without New Technology Among Romanian High School Students“. European Journal of Interdisciplinary Studies 3, Nr. 3 (19.05.2017): 22. http://dx.doi.org/10.26417/ejis.v3i3.p22-30.
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Nowadays, the new technology defines the classroom and students’ and teachers’ life. Sometimes the attitude towards technology use is marked by negative dysfunctional emotion, anxiety, fear, avoidance or dependence. This paper focuses both on the anxiety with the new technology and on the anxiety without technology. Our research aims to examine the attitudes and behaviours of Romanian high school students regarding trendy technologies, such as computers, internet and smartphones, including the access to social networking applications. The following tools were used: CARS (Heinssen, Glass, - Knight, 1987), IAS (Nickel and Pinto, 1986), four scales from MTUAS (Rosen, Whaling, Carrier, Cheever, and Rokkum, 2013), the Use of Smartphones for Learning Purposes Scale-USLS and a socio-demographic questionnaire. There were 517 participants distributed in two studies. The findings showed some differences concerning gender, age, specialization and academic performance, as well as an evolution of participants from the anxiety towards the computer (highest with females) to the anxiety without technology (similar for females and males). School performance is negatively associated with computer anxiety and Facebook activities. The study is important in the Romanian context, where computers, internet and smartphone penetration is more pronounced with younger people. Pedagogical issues of the research are also discussed, anxiety having a double function, as an endogenous and exogenous factor with respect to one’s academic and professional development.
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Cocoradă, Elena. „Anxiety with and Without New Technology Among Romanian High School Students“. European Journal of Interdisciplinary Studies 8, Nr. 1 (19.05.2017): 22. http://dx.doi.org/10.26417/ejis.v8i1.p22-30.
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Nowadays, the new technology defines the classroom and students’ and teachers’ life. Sometimes the attitude towards technology use is marked by negative dysfunctional emotion, anxiety, fear, avoidance or dependence. This paper focuses both on the anxiety with the new technology and on the anxiety without technology. Our research aims to examine the attitudes and behaviours of Romanian high school students regarding trendy technologies, such as computers, internet and smartphones, including the access to social networking applications. The following tools were used: CARS (Heinssen, Glass, - Knight, 1987), IAS (Nickel and Pinto, 1986), four scales from MTUAS (Rosen, Whaling, Carrier, Cheever, and Rokkum, 2013), the Use of Smartphones for Learning Purposes Scale-USLS and a socio-demographic questionnaire. There were 517 participants distributed in two studies. The findings showed some differences concerning gender, age, specialization and academic performance, as well as an evolution of participants from the anxiety towards the computer (highest with females) to the anxiety without technology (similar for females and males). School performance is negatively associated with computer anxiety and Facebook activities. The study is important in the Romanian context, where computers, internet and smartphone penetration is more pronounced with younger people. Pedagogical issues of the research are also discussed, anxiety having a double function, as an endogenous and exogenous factor with respect to one’s academic and professional development.
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Contreras-Jurado, Constanza, Laura García-Serrano, Mariana Gómez-Ferrería, Clotilde Costa, Jesús M. Paramio und Ana Aranda. „The Thyroid Hormone Receptors as Modulators of Skin Proliferation and Inflammation“. Journal of Biological Chemistry 286, Nr. 27 (12.05.2011): 24079–88. http://dx.doi.org/10.1074/jbc.m111.218487.
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We have analyzed the role of the thyroid hormone receptors (TRs) in epidermal homeostasis. Reduced keratinocyte proliferation is found in interfollicular epidermis of mice lacking the thyroid hormone binding isoforms TRα1 and TRβ (KO mice). Similar results were obtained in hypothyroid animals, showing the important role of the liganded TRs in epidermal proliferation. In addition, KO and hypothyroid animals display decreased hyperplasia in response to 12-O-tetradecanolyphorbol-13-acetate. Both receptor isoforms play overlapping functional roles in the skin because mice lacking individually TRα1 or TRβ also present a proliferative defect but not as marked as that found in double KO mice. Defective proliferation in KO mice is associated with reduction of cyclin D1 expression and up-regulation of the cyclin-dependent kinase inhibitors p19 and p27. Paradoxically, ERK and AKT activity and expression of downstream targets, such as AP-1 components, are increased in KO mice. Increased p65/NF-κB and STAT3 phosphorylation and, as a consequence, augmented expression of chemokines and proinflammatory cytokines is also found in these animals. These results show that thyroid hormones and their receptors are important mediators of skin proliferation and demonstrate that TRs act as endogenous inhibitors of skin inflammation, most likely due to interference with AP-1, NF-κB, and STAT3 activation.
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Guo, Luming, Xiaoling Xie, Jing Wang, Haiyan Xiao, Shuchun Li, Mei Xu, Ebenezer Quainoo et al. „Inducible Rbpms-CreERT2 Mouse Line for Studying Gene Function in Retinal Ganglion Cell Physiology and Disease“. Cells 12, Nr. 15 (27.07.2023): 1951. http://dx.doi.org/10.3390/cells12151951.
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Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain, and dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing for the co-expression of RBPMS and CreERT2. The inducible Rbpms-CreERT2 mice exhibited a high recombination efficiency in activating the expression of the tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, both heterozygous and homozygous Rbpms-CreERT2 knock-in mice showed no detectable defect in the retinal structure, visual function, and transcriptome. Together, these results demonstrated that the Rbpms-CreERT2 knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study, and ocular disease modeling in RGCs.
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Ricketts, M. H., und A. D. Levinson. „High-level expression of c-H-ras1 fails to fully transform rat-1 cells“. Molecular and Cellular Biology 8, Nr. 4 (April 1988): 1460–68. http://dx.doi.org/10.1128/mcb.8.4.1460-1468.1988.
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Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.
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Suwannakot, Kornrawee, Nataya Sritawan, Salinee Naewla, Anusara Aranarochana, Apiwat Sirichoat, Wanassanun Pannangrong, Peter Wigmore und Jariya Umka Welbat. „Melatonin Attenuates Methotrexate-Induced Reduction of Antioxidant Activity Related to Decreases of Neurogenesis in Adult Rat Hippocampus and Prefrontal Cortex“. Oxidative Medicine and Cellular Longevity 2022 (15.07.2022): 1–13. http://dx.doi.org/10.1155/2022/1596362.
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Previous studies have revealed that the side effects of anticancer drugs induce a decrease of neurogenesis. Methotrexate (MTX), one of anticancer drugs, can induce lipid peroxidation as an indicator of oxidative stress in the brain. Melatonin has been presented as an antioxidant that can prevent oxidative stress-induced neuronal damage via the activation of antioxidant enzymes associated with the increase of neurogenesis. The aims of the present study are to examine the neuroprotective effect of melatonin on the neurotoxicity of MTX on neurogenesis and the changes of protein expression and antioxidant enzyme levels in adult rat hippocampus and prefrontal cortex (PFC). Male Sprague-Dawley rats were assigned into four groups: vehicle, MTX, melatonin, and melatonin+MTX groups. The vehicle group received saline solution and 10% ethanol solution, whereas the experimental groups received MTX (75 mg/kg, i.v.) and melatonin (8 mg/kg, i.p.) treatments. After the animal examination, the brains were removed for p21 immunofluorescence staining. The hippocampus and PFC were harvested for Western blot analysis and biochemical assessments of malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD). The immunofluorescence result showed that coadministration with melatonin diminished p21-positive cells in the hippocampal dentate gyrus, indicating a decrease of cell cycle arrest. Melatonin reduced the levels of MDA and prevented the decline of antioxidant enzyme activities in rats receiving MTX. In the melatonin+MTX group, the protein expression results showed that melatonin treatment significantly upregulated synaptic plasticity and an immature neuron marker through enhancing brain derived neurotrophic factor (BDNF) and doublecortin (DCX), respectively. Moreover, melatonin ameliorated the antioxidant defense system by improving the nuclear factor erythroid 2-related factor 2 (Nrf2) in rats receiving MTX. These findings suggested that the effects of melatonin can ameliorate MTX toxicity by several mechanisms, including an increase of endogenous antioxidants and neurogenesis in adult rat hippocampus and PFC.
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Ricketts, M. H., und A. D. Levinson. „High-level expression of c-H-ras1 fails to fully transform rat-1 cells.“ Molecular and Cellular Biology 8, Nr. 4 (April 1988): 1460–68. http://dx.doi.org/10.1128/mcb.8.4.1460.
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Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.
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Pilunov, Artem, Dmitrii Romaniuk, Savely Sheetikov, Alexandra Khmelevskaya, Anton Shmelev, Nadia Bykova, Alina Shomuradova et al. „Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1“. Blood 134, Supplement_1 (13.11.2019): 5749. http://dx.doi.org/10.1182/blood-2019-130552.
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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance <3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.
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Crozier, Stephen J., Maria Dolors Sans, Charles H. Lang, Louis G. D'Alecy, Stephen A. Ernst und John A. Williams. „CCK-induced pancreatic growth is not limited by mitogenic capacity in mice“. American Journal of Physiology-Gastrointestinal and Liver Physiology 294, Nr. 5 (Mai 2008): G1148—G1157. http://dx.doi.org/10.1152/ajpgi.00426.2007.
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In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.
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Zhang, Shengliang, und Wafik S. El-Deiry. „Abstract 2604: Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer cells“. Cancer Research 83, Nr. 7_Supplement (04.04.2023): 2604. http://dx.doi.org/10.1158/1538-7445.am2023-2604.
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Abstract The p53 codon p72 polymorphism with proline (p53P72)/arginine (p53R72) substitution is associated with cancer incidence. Cysteine(C) substitution at p53 codon 72 polymorphism (p53C72) is rarely studied in cancer patients. A patient who developed AML and who carries p53C72 was described (El-Deiry, 2022 WIN Symposium). The patient’s family has high rates of cancer susceptibility in the women some of whom carry FANCC alterations and other alleles. To examine the functional characteristics of different p53 alterations at codon 72, we generated plasmids expressing p53C72, p53 R72 or p53P72. The variants of p53 were overexpressed in p53-null cancer cells by transient transfection of the plasmids. We tested functions of the p53 codon 72 polymorphisms (p53C72, p53R72 and p53P72) in two cancer cell lines (Soas-2 and HCT116 p53 null) with different expression levels of the p53 variants at different time points. To examine the effect of codon 73 variants on p53 transactivation, we performed a PG13-Luciferase reporter assay and examined the endogenous p53 targets at the protein level. The luciferase-reporter assay showed that all the three variants of the p53 codon p72 variants increased PG-13 luciferase expression at early time points. The Western blot assay showed that the p53C72, the p53R73 and the p53P72 increased endogenous p21, DR5, puma and MDM2 at the protein levels at different time points. The p53C72 increased the p53 targets and PG13-driven luciferase expression much higher than the p53R72 and the p53P72. These results suggest that the p53 codon 72 variants (C72, R72 and P72) retain wild-type activity for p53-mediated transcription, and p53C72 has the highest potency among them. We examined the effect of the p53 variants on cell growth and death. PARP cleavage (a marker of cell apoptosis) was induced in cells overexpressing all the three p53 codon 72 variants. We observed a lesser potency of p53C72 to induce PARP cleavage compared to p53R72 and p53P72. Colony formation assay showed a suppressive effect of p53C72, p53R72 and p53P72 on cancer cell growth. Our studies demonstrate some differences of in wild-type functions of p53 p53C72, p53R72 and p53P72 alleles. Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2604.
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Vega, Francisco, L. Jeffrey Medeiros, Coralyn Atwell, Jeong Hee Cho, Ling Tian, Francois-Xavier Claret und George Z. Rassidakis. „Activation of mTOR Signaling Pathway Contributes to Tumoral Cell Survival in ALK-Positive Anaplastic Large Cell Lymphoma.“ Blood 106, Nr. 11 (16.11.2005): 2419. http://dx.doi.org/10.1182/blood.v106.11.2419.2419.
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Abstract Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of nucleophosmin (NPM)-ALK. Previously, NPM-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. Recently, we have shown that mTOR signaling proteins are activated in ALK-positive ALCL tumors and that mTOR activation depends, at least in part, on activation of AKT (Lab Invest2005; 85: 255A). In this study, we investigate the biological effects of inhibition of mTOR on two ALK-positive ALCL cell lines, Karpas 299 and SU-DHL1. For this purpose, we used rapamycin to inhibit mTOR-raptor complex and mTOR-specific small interfering RNA (siRNA) to silence the endogenous mtor gene. Treatment with rapamycin, resulted in a marked concentration-dependent decrease of phosphorylated (p)-mTOR, and its downstream targets, p-p70S6K, p-S6K, p-4E-BP1 and total eIF4E. Similarly, silencing the expression of mtor resulted in a decrease in the activation/phosphorylation level of these proteins as well as in the level of p-AKT. Both treatments induced apoptosis and cell cycle arrest in both ALK-positive ALCL cell lines as demonstrated by trypan blue exclusion, annexin V staining, BrdU incorporation, and cell cycle studies. There was a concentration-dependent decrease in the anti-apoptotic proteins BCL-2, BCL-XL, MCL-1 and c-FLIP (L and S) with increasing concentrations of rapamycin or after mTOR siRNA treatment. The cyclin dependent kinase inhibitors p21waf1 and p27kip1 and underphosphorylated (Un-p)-RB protein were upregulated, after treatment with rapamycin or after mTOR siRNA treatment. In conclusion, we provide evidence that inhibition of mTOR induces cell cycle arrest and apoptosis in ALK-positive ALCL cells. The decrease of p-AKT by silencing mtor suggests that mTOR is necessary to activate AKT in ALK-positive ALCL, and thus, mTOR can function as a feedback signal activity of its own pathway.
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Liu, W., Q. LI, X. Han, L. Sun und J. Wang. „POS0413 IGURATIMOD AMELIORATES BLEOMYCIN-INDUCED PULMONARY FIBROSIS VIA INHIBITINGEMT PROCESS AND NLRP3 INFLAMMASOME ACTIVATION“. Annals of the Rheumatic Diseases 81, Suppl 1 (23.05.2022): 462.3–462. http://dx.doi.org/10.1136/annrheumdis-2022-eular.969.
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BackgroundInterstitial pulmonary fibrosis is the deadliest manifestation of rheumatoid arthritis (RA), and its pathogenesis is complicated. As a new drug for controlling RA, clinical studies have found that iguratimod (IGU) has certain advantages in improving lung function and shows great potential for pulmonary fibrosis therapy[1]. However, the specific mechanism of IGU in treating pulmonary fibrosis is not clear.ObjectivesThis study was designed to observe and investigate the therapeutic effects of IGU on bleomycin-induced pulmonary fibrosis in mice and further investigate its underlying mechanism, aiming to provide a further theoretical basis for clinical rational drug use.MethodsA mouse model of pulmonary fibrosis was induced by intratracheal injection of bleomycin (BLM)[2]. Model mice were randomly assigned to receive Sodium carboxymethyl cellulose (CMC) or different concentrations of IGU. HE staining and immunohistochemical staining were performed to observe the therapeutic effects of IGU on mouse fibrosis. TGF-β induced A549 EMT cell model was utilized to investigate the effects of IGU on EMT in vitro[3]. NLRP3 inflammasome was activated by the co-stimulation of TGF-β+LPS+ATP (TLA) to evaluate the effects of IGU in vitro[4].ResultsWe found that IGU resulted in favorable therapeutic outcomes by affecting the inflammation infiltration (Figure 1A) and collagen deposition (Figure 1B). Immunohistochemical results showed that IGU downregulated the levels of α-SM and NLRP3. Additionally, the markers of BLM-mediated EMT (Figure 1C) phenotype and NLRP3-activated (Figure 1D) phenotype in the lung were also attenuated after IGU administration. In vitro experiments, the results confirmed its anti-EMT effects of reducing the expression of interstitial proteins Vimentin and α-SMA and restoring the content of epithelial protein E-cadherin. Besides, IGU could inhibit NLRP3 activation by downregulating NLRP3 related marker proteins, reducing the secretion of IL-1β, and attenuating caspase-1 activity. We then found that IGU anti-EMT and anti-NLRP3 effect was accompanied by decreased ROS production.Figure 1.IGU ameliorates BLM-induced pulmonary inflammation and pulmonary fibrosis. (A) Representative lung micrographs of lung tissues stained with HE. Magnification, × 200. (B) Representative photographs of lung tissues stained with Masson’s trichrome staining. Magnification, × 200. (C) Representative results of western blot for E-cadherin, Vimentin, α-SMA expression in lung tissues. GAPDH was used as the endogenous control. (D) Representative results of western blot for NLRP3, caspase-1, cleaved-caspase-1 p20, IL-1β expression in lung tissues. GAPDH was used as the endogenous control.ConclusionIGU can inhibit the EMT process and NLRP3 inflammasome activation, reduce ROS production to ameliorate pulmonary fibrosis, which may provide new insights into the further application of IGU in Interstitial pulmonary fibrosis.References[1]Shu P, et al. Eur Rev Med Pharmacol Sci 2021, 25(4):4687 v Med[2]Philipp K, et al. Eur Respir J 2020, 55(6):1901105.[3]Justin C. H, et al. Matrix Biol 2018, 71-72:112-127.[4]Anita A P, et al. Front Pharmacol 2020, 11:1201.Disclosure of InterestsNone declared.
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Peery, Robert Craig, Jiejun Shi, Leah Farmer, Li Yang, Emily Lawrence, Yumei Li, Wei Li und Lanlan Shen. „Abstract 6016: A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells“. Cancer Research 83, Nr. 7_Supplement (04.04.2023): 6016. http://dx.doi.org/10.1158/1538-7445.am2023-6016.
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Abstract Background: Gut microbial colonization, represents a critical time period for establishing the overall health of an individual. The intestine is a highly dynamic tissue with high turnover of intestinal epithelial cells throughout the life span of an individual making intestinal stem cells (ISCs) critical for normal gut homeostasis. Currently, there exists a lack of knowledge in how the gut microbiota regulates DNA methylation to influence the behavior of these ISCs. Aberrant DNA methylation is now documented to be critical in the disease pathogenesis of intestinal disorders such as colorectal cancer, thus understanding how the gut microbiome influences DNA methylation and ISCs is fundamental for improving human health. Methods: We utilized the Lgr5-GFP mouse model in which endogenous GFP expression is driven by the bonafide intestinal stem cell marker Lgr5. These mice were maintained under two different housing conditions, conventional and germ free. We collected mice under the two conditions and performed dissociation of the colon and small intestine epithelial cells using EDTA dissociation. We then analyzed cell populations using flow cytometry, and collected two cell populations: the differentiated cell population (EpiCam +, GFP -) and intestinal stem cell population (EpiCam +, GFP +). To perform a genome wide comprehensive DNA methylation analysis we performed Whole Genome Bisulfite Sequencing (WGBS) which included both stem cell and differentiated cell populations of females and males at the two age groups points (postnatal day 21 (p21) and p100). Results: There was no significant difference in CpG methylation at the global level between the two housing conditions. Next, we analyzed age matched samples from germ free and conventional mice to determine if there are differentially methylated regions (DMRs) associated with the presence of intestinal microbiota. We have identified DMRs that are maintained in germ free stem cells from p21 to p100 compared to conventional mice, as well as DMRs in the differentiated cell population at both ages. Interestingly in our analysis several genes displayed robust hypomethylation of control mice versus germ free mice at the p100 time point in both the stem cell and differentiated cell populations. One such gene, Ifitm3, has been heavily associated with chronic inflammation in the colonic epithelium and a poor risk factor for colonic cancer (Li D et. Al, Clin Cancer Res. 2011). We validated Ifitm3 methylation and expression changes between cohorts using pyrosequencing and RT-PCR respectively. Conclusions: This comprehensive analysis has yielded top candidate genes for studying functional consequences of DNA methylation changes that have been previously linked to severe patient diseases including cancer. Citation Format: Robert Craig Peery, Jiejun Shi, Leah Farmer, Li Yang, Emily Lawrence, Yumei Li, Wei Li, Lanlan Shen. A comprehensive analysis of gut microbiome mediated epigenetic regulation of intestinal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6016.
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Oh, Hyeon Jeong, Seah Park, Hong-Kyung Yang, Hoon Ryu, Young-Joon Surh, Dae-Yong Kim und Hye-Kyung Na. „Abstract 5252: Salsolinol suppresses the STAT3-EMT axis in liver carcinogenesis“. Cancer Research 83, Nr. 7_Supplement (04.04.2023): 5252. http://dx.doi.org/10.1158/1538-7445.am2023-5252.
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Abstract Liver cancer is one of the most common malignancies and a leading cause of death worldwide. However, it is still very difficult to treat and prevent liver cancer. Salsolinol (SAL) is an endogenous catechol isoquinoline generated by the condensation of dopamine with acetaldehyde, a major metabolite of ethanol. In the present study, we found that SAL inhibited the phosphorylation, dimerization, and subsequent nuclear translocation of STAT3 in a concentration-dependent manner in SK-Hep1 cells, a hepatic carcinoma cell line. SAL suppressed the expression of Cdk 4 and Cyclin D1, the major target protein of STAT3, and induced the cell cycle arrest at Go/G1 phase. However, SAL induced the expression of STAT1 in SK-Hep1 cells. In addition, SAL enhanced the expression of the cell cycle regulators such as p53 and p21 in SK-Hep1 cells. Epithelial-mesenchymal transition (EMT) promotes tumor invasion and metastasis in malignancies through STAT3 activation. SAL induced the expression of E-cadherin while suppressing the expression of Snail, N-cadherin, and Slug, which are related to EMT. SAL inhibited the migration of SK-Hep1 cells by suppressing the expression of MMP-9 and MMP-2. Furthermore, intraperitoneal injection of SAL reduced the number of tumors in the diethylnitrosamine (DEN)-induced liver carcinogenesis model without affecting the body weight change. Proliferative markers PCNA and KI-67 and p-STAT3 were down-regulated in the SAL-injected mice liver cancer tissue. The plasma level of alpha-fetoprotein, a tumor marker in the blood, was also reduced in the SAL-injected mice. Interestingly, SAL injection significantly reduced the anxiety-like behavior without affecting the locomotive activities in the DEN-induced hepatocellular carcinoma mouse model. Moreover, SAL injection recovered tyrosine hydroxylase, a key enzyme for producing dopamine from tyrosine in the substantia nigra compacta of DEN-treated mice. Together, SAL inhibits STAT3 signaling, thereby suppressing the cell cycle progression and EMT in SK-Hep1 cells, which may account for its anti-depressant and anti-carcinogenic activity in DEN-induced liver carcinogenesis. Citation Format: Hyeon Jeong Oh, Seah Park, Hong-Kyung Yang, Hoon Ryu, Young-Joon Surh, Dae-Yong Kim, Hye-Kyung Na. Salsolinol suppresses the STAT3-EMT axis in liver carcinogenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5252.
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Qian, Hong, und Mikael Sigvardsson. „Early B-Cell Factor 2 Represents A Novel Marker of Highly Purified Messenchymal Stem-Like Cells in Mouse Bone Marrow.“ Blood 114, Nr. 22 (20.11.2009): 252. http://dx.doi.org/10.1182/blood.v114.22.252.252.
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Abstract Abstract 252 The future therapeutic use of mesenchymal stem cells (MSCs) for human depends on the establishment of preclinical studies with other mammals such as mouse. However, purification and characterization of mouse MSCs from bone marrow (BM) remain poorly documented. The lack of MSC-specific markers for isolation and characterization has been a main obstacle to both research and clinical application with MSCs. The isolation method based on the adherence properties of MSCs in culture has proven ineffective because of large contamination of various hematopoietic lineages and potential phenotypic changes in cultured cells. Consequently, there is very little knowledge about the precise properties of a MSC in its native environment. In the present study, we have utilized a bacterial artificial chromosomes (BAC) transgenic reporter mouse line expressing enhanced green fluorescent protein (EGFP) under the control of the regulatory elements of the Ebf2 gene. Early B cell factor 2 (EBF2) , is a member of the EBF family of transcription factors and has been shown to be expressed in the endosteal niche (Kieslinger et al 2005), a region where MSCs may be defined. The Ebf2-EGFP expressing stromal cells (CD45−TER119−GFP+) are composed 0.002% of total BM mononuclear cells and could be sorted by fluorescence-activated cell sorter (FACS) from adult Tg (Ebf2-EGFP)FB58Gsat/Mmcd mice. The fidelity of GFP expression to that of the endogenous Ebf2 gene was confirmed by quantitative real time PCR providing evidences for that the reporter gene expression marked a defined population of CD45− cells. GFP+ cells could not be found in the hematopoietic cell compartments (CD45+TER119+), indicating a unique expression of EBF2 in stromal cells. In addition, a 10-fold reduction in frequency of CD45−TER119−GFP+ cells in marrow cells compared to that in bone suggested a preferential distribution of the GFP+ stromal cells in endosteal area of the bone. Colony forming unit-fibroblast (CFU-F) assay of the sorted cells revealed that the frequency of CFU-Fs in CD45−TER119−GFP+ cells reached as high as 1 out of 15 whereas only around1/4000 could be detected in CD45−TER119−GFP− cells, indicating the GFP+ EBF2 expressing cells are enriched for primitive stromal progenitor cells in BM. Morphologically, CFU-Fs derived from the GFP+ cells are mostly spindle-shaped and mononuclear. In contrast, the CFU-Fs derived from the GFP- cells are enriched with big cells containing multiple nuclei and differentiated stromal cells. In line with the function and morphological data, Microarray data obtained from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca2-4, −7,−8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the GFP+ cells, compared to the GFP− cells, indicating quiescence state of GFP+ cells. Even though the GFP+ cells functionally appeared more primitive than the GFP− cells, multiple lineage associated genes specific for osteoblasts, adipocyte, chondrocyte and myocyte were relatively higher expressed in the GFP+ cell population compared to the GFP− cells, possibly indicating lineage priming events. To test the expression profiles of cell surface antigens that has been studied in the MSCs selected in culture, we performed multiple-color FACS analysis of expressions of CD34, SCA1, CD44 and CD29 within CD45−TER119−GFP+ cells. In order to exclude contamination of hematopoietic cells, we add additional markers B220/CD19 in separated channels during the FACS analysis. Consistent with the Microarray data, we found that GFP+ cells are 100% CD29+, but 100% CD44−. In addition, they express higher levels of CD34, SCA1, compared to the GFP− cells. This is in contrast to the previous studies showing that the MSCs from culture express higher level of CD44 and most of them are CD34−. Thus, using the transgenic EBF2 reporter mouse model we have been able to prospectively isolate an MSC like cell directly from the adult mouse BM largely increasing the possibilities to investigate phenotypic and molecular characteristics of the BM primitive mesenchymal progenitor cells ex vivo. Disclosures: No relevant conflicts of interest to declare.
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Suzuki, Naoya, Asuka Hira, Akira Niwa, Megumu Saito, Keitaro Matsuo, Tatsutoshi Nakahata, Minoru Takata und Miharu Yabe. „Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity“. Blood 120, Nr. 21 (16.11.2012): 648. http://dx.doi.org/10.1182/blood.v120.21.648.648.
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Abstract Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures: No relevant conflicts of interest to declare.
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Kurlekar, Samvid, Joanna D. C. Lima, Chris W. Pugh, Julie Adam und Peter J. Ratcliffe. „Abstract 862: Negative cellular outcomes following acute in vivo Vhl inactivation in mice“. Cancer Research 82, Nr. 12_Supplement (15.06.2022): 862. http://dx.doi.org/10.1158/1538-7445.am2022-862.
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Abstract Clear cell renal cell carcinoma (ccRCC) is driven by the biallelic inactivation of the von Hippel Lindau tumor suppressor (VHL) gene, which results directly in the normoxic stabilization of hypoxia inducible factors (HIF-1 and -2). While VHL is expressed ubiquitously, its inactivation is tumorigenic only in specific tissues. We hypothesize that this tissue-specificity arises from a negative selective pressure on VHL KO cells that is tolerated in certain contexts. To assess this phenomenon, we induced Vhl deletion in vivo simultaneously across the mouse body and observed cell fate with single-cell resolution. Here we show that inducing widespread inter-organ Vhl deletion results in a massive removal of affected cells from tissue. Our study required technology that allowed us to visualize and isolate Vhl-recombined cells from tissue. For this, we developed a novel lineage marking model in which Cre-mediated excision of Vhl is coupled to knock-in of a tdTomato reporter cassette under the endogenous Vhl promoter. We used this model in conjunction with the ubiquitously-expressed UBC-CreERT2 to induce marked biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl. One week after inactivation was induced by administration of tamoxifen, there were equal numbers of tdTomato-positive cells in both genotypes, implying that initial recombination was similar and that Vhl KO did not affect immediate cell survival. Surprisingly, there was a massive reduction in the number of tdTomato-positive cells in multiple KO/KO organs after four weeks. This loss was accompanied by proliferation of tdTomato-negative cells, as assayed by BrdU incorporation, suggesting that tissues can at least partially compensate for loss of Vhl-inactivated cells. Finally, we found that the removal of cells was preceded by signs of cellular damage and stress, such as accumulation of p27, cytoplasmic translocation of HMGB1, and TUNEL-positivity in the kidney and liver. Taken together, we conclude that Vhl inactivation is inherently deleterious to cells and entrains a negative cell selection process in multiple organs. One corollary is that there must exist some conditions under which such selection might not occur; secondary mutations found in ccRCC, such as in Pbrm1, or loss of individual HIF isoforms, might provide such conditions to Vhl KO cells. Currently we are investigating the role of these additional perturbations in modifying the cellular response to Vhl loss. Our study will provide new insights into the genesis of ccRCC by redefining the mechanisms underlying the creation of pro-tumorigenic niches following Vhl inactivation. Citation Format: Samvid Kurlekar, Joanna D C Lima, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. Negative cellular outcomes following acute in vivo Vhl inactivation in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 862.
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Cho, Hearn J., Anna Mei, Tricia Nardiello, Maurizio DiLiberto, Xiangao Huang, Jennifer E. Amengual, Achim Jungbluth et al. „MAGE-A3 Inhibits p53 and Promotes Proliferation and Survival in Multiple Myeloma.“ Blood 114, Nr. 22 (20.11.2009): 1795. http://dx.doi.org/10.1182/blood.v114.22.1795.1795.
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Abstract Abstract 1795 Poster Board I-821 The type I Melanoma Antigen GEne (MAGE) proteins MAGE-A3 and CT7 (MAGE-C1) were commonly detected in primary tumor cells from multiple myeloma patients and their expression was correlated with advanced disease and proliferation. They belong to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome. These findings suggest that type I MAGE may play a pathogenic role in proliferation or survival in multiple myeloma cells. To test this hypothesis, we examined MAGE expression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). First, we examined CTAg expression and proliferation in vivo at two critical clinical milestones, in newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003), whereas CT7 was detected in about 75% of both patient populations. The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating an association between MAGE-A3, progression of disease and proliferation. Second, we investigated the functional role of MAGE-A3 by silencing this gene in HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA (≥90% compared to controls) and protein in MM.1r and Arp-1 HMCL by 48 hours and this effect was maintained up to 96 hours. Pulse labeling of HMCL with bromodeoxyuridine for 30 minutes revealed that silencing of MAGE-A3 led to cell cycle arrest, as evidenced by the complete loss of cells in S phase and accumulation of cells in both G1 and G2. This was accompanied by increased expression of the tumor suppressor p53 and the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, a p53 target that inhibits CDKs in both late G1 and G2. However, CDK4/6-specific phosphorylation of the retinoblastoma gene product (Rb) was unimpaired, indicating that control of the mid-G1 cell cycle checkpoints by Rb remained intact and suggesting that MAGE-A3 acted in part to promote G1-S progression. Within 24 hours of cell cycle arrest, 70-80% of MAGE-A3-silenced cells underwent apoptosis as measured by Annexin V staining, compared to '20% in cells transduced with a non-target control lentivirus or untreated. Furthermore, this apoptosis was caspase-dependent, as it was completely prevented by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh, and was triggered by the loss of mitochondrial outer membrane potential in the activation of the intrinsic apoptosis pathway. Taken together, the in vivo and in vitro results suggest that MAGE-A3 promoted myeloma cell proliferation by inhibiting p53-dependent expression of p21, and loss of this activity leads to growth arrest and cell cycle-coupled apoptosis via activation of the intrinsic apoptosis pathway. Understanding the biochemical mechanism of MAGE-A3 in cell cycle regulation and survival may identify novel therapeutic strategies for multiple myeloma. Proof of principle in this disease may lead to broader application of these strategies in other cancers that express MAGE-A3. Disclosures Niesvizky: Proteolix: Research Funding, data monitoring committee; Seattle Genetics, Inc: Research Funding; Celgene: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau.
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Grasshoff, H., S. Comduehr, J. Ohmes, D. Thaçi, K. Schulze-Forster, H. Heidecke, P. Lamprecht und G. Riemekasten. „POS0875 AUTOANTIBODIES DIRECTED TO G-PROTEIN COUPLED RECEPTORS CORRELATE WITH DISEASE ACTIVITY SCORES IN PATIENTS WITH PSORIATIC ARTHRITIS“. Annals of the Rheumatic Diseases 82, Suppl 1 (30.05.2023): 743.2–744. http://dx.doi.org/10.1136/annrheumdis-2023-eular.6068.
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BackgroundPsoriatic arthritis (PsA) is a chronic inflammatory disease that affects the skin, nails, tendons and joints. In addition, several comorbidities have been identified with PsA. In particular, a frequent occurrence of fatigue and mental disorders such as depression have been described in patients with PsA. Key cellular players in pathophysiology of PsA are T cells. However, in recent years, various autoantibodies such as anti-LL37 and anti-Pso-p27 have been identified in PsA as well [1]. Autoantibodies targeting G protein-coupled receptors (GPCRs) have been identified in several diseases and are involved in disease pathophysiology [2]. Many of these autoantibodies are agonists that enhance the effect of the natural ligand at the receptor [3].ObjectivesThe aim of this study was to investigate the association between autoantibodies directed to distinct GPCRs and disease activity as well as comorbidities in PsA.MethodsIn a prospective observational study forty-five patients with an active PsA according to CASPAR criteria and 24 healthy controls (HCs) matched according to age and sex were included in this study. The serum-concentrations of 20 autoantibodies directed to GPCRs involved in the renin-angiotensin-aldosterone system, the sympathetic and parasympathetic nervous system as well as chemokine, complement and scavenger receptors, were measured by the company CellTrend GmbH (Luckenwalde, Germany). Autoantibodies measured in patient’s sera at baseline were transformed and adjusted for age as confounder for further analyses. To compare autoantibody concentrations between PsA and HCs we applied Two-Way-ANOVA. Correlation analyses were performed using Pearson correlation. Autoantibody concentrations between patients with and without depression were analyzed by Student’s T test and binary logistic regression.ResultsPatients were equally distributed according to sex (male 51.1%, female 48.9%) and of a mean age of 47.5 ± 13.2 years. Mean arthritic disease manifestation assessed by DAPSA was 44.4 ± 21.2. Patients displayed a disease activity with a mean PASI of 6.19 ± 6.33, and high impact on patient reported outcomes with a mean DLQI of 10.3 ± 7.4 and a mean HAQ of 1.0 ± 0.8. Autoantibody concentrations did not differ significantly between patients with PsA and HCs. Further analysis revealed significant correlations between autoantibody levels, disease activity scores and treatment response of cutaneous disease manifestation after 16 weeks of treatment (Δ PASI),Figure 1. Moreover, the levels autoantibodies directed to the MAS receptor, CXCR3, PAR1 and stabilin were significantly higher in patients with PsA suffering from depression compared to patients without depression (MASR: p=0.035; CXCR3: p=0.011; PAR1: p=0.047; stabilin: p=0.035), although binary logistic regression did not prove any predictive value for these four antibodies.ConclusionAutoantibodies directed to GPCRs correlated with disease activity scores in patients with PsA. Further larger analyses regarding the functional role of these autoantibodies in PsA are required.References[1]Koussiouris J, Chandran V. Autoantibodies in Psoriatic Disease. The Journal of Applied Laboratory Medicine (2022) 7:281–293. doi: 10.1093/jalm/jfab120[2]Skiba MA, Kruse AC. Autoantibodies as Endogenous Modulators of GPCR Signaling. Trends in Pharmacological Sciences (2021) 42:135–150. doi: 10.1016/j.tips.2020.11.013[3]Yue X, Yin J, Wang X, Heidecke H, Hackel AM, Dong X, Kasper B, Wen L, Zhang L, Schulze-Forster K, et al. Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching. Ann Rheum Dis (2022)annrheumdis-2021-222088. doi: 10.1136/annrheumdis-2021-222088Figure 1.Heatmap displaying Pearson’s correlation coefficient of correlation analyses between diseases activity scores and autoantibody concentrations adjusted for age at baseline. Pearson correlation analyses with a p-value <0.05 are marked with a box.Acknowledgements:NIL.Disclosure of InterestsHanna Grasshoff Speakers bureau: Abbvie, Sara Comduehr: None declared, Justus Ohmes: None declared, Diamant Thaçi Speakers bureau: Diamant Thaçi has received honoraria as an advisor, speaker and/or investigator from AbbVie, Almirall, Amgen, Biogen-Idec, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Galapagos, Galderma, Janssen-Cilag, LEO Pharma, Novartis, Pfizer, Regeneron, Roche-Possay, Samsung, Sanofi and UCB., Consultant of: Diamant Thaçi has received honoraria as an advisor, speaker and/or investigator from AbbVie, Almirall, Amgen, Biogen-Idec, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Galapagos, Galderma, Janssen-Cilag, LEO Pharma, Novartis, Pfizer, Regeneron, Roche-Possay, Samsung, Sanofi and UCB, Kai Schulze-Forster Shareholder of: board of management: CellTrend, Harald Heidecke Shareholder of: board of management: CellTrend, Peter Lamprecht: None declared, Gabriela Riemekasten: None declare.d.
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Zheng, Boshen, Yue Fan, Wei Wei, Yourui Xu, Shaowei Huang und Shengwei Mei. „Distribution Optimal Power Flow With Energy Sharing Via a Peer-To-Peer Transactive Market“. Frontiers in Energy Research 9 (15.07.2021). http://dx.doi.org/10.3389/fenrg.2021.701149.
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The technology advancement and cost decline of renewable and sustainable energy increase the penetration of distributed energy resources (DERs) in distribution systems. Transactive energy helps balance the local generation and demand. Peer-to-peer (P2P) energy trading is a promising business model for transactive energy. Such a market scheme can increase the revenue of DER owners and reduce the waste of renewable energy. This article proposes an equilibrium model of a P2P transactive energy market. Every participant seeks the maximum personal interest, with the options of importing or providing energy from/to any other peer across different buses of the distribution network. The market equilibrium condition is obtained by combining the Karush–Kuhn–Tucker conditions of all problems of individual participants together. The energy transaction price is endogenously determined from the market equilibrium condition, which is cast as a mixed-integer linear program and solved by a commercial solver. The transactive energy flow is further embedded in the optimal power flow problem to ensure operating constraints of the distribution network. We propose a remedy to recover a near optimal solution when the second-order cone relaxation is inexact. Finally, a case study demonstrates that the proposed P2P market benefits all participants.
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Zennyo, Yusuke. „Product variety and design in the age of peer‐to‐peer sharing“. Journal of Economics & Management Strategy, 06.11.2023. http://dx.doi.org/10.1111/jems.12563.
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AbstractThe rise of peer‐to‐peer (P2P) sharing, exemplified recently by increased car‐sharing and clothing‐sharing, has altered our consumption style. One can consume goods without owning them. In fact, the ownership of goods can be monetized through P2P rental markets. These changes are regarded as influencing various strategies of manufacturers of goods being shared. Specifically, this paper examines aspects of product variety and design. A stylized model is examined in which a manufacturing firm makes a product variety decision of whether to launch a niche product line in addition to an existing mass product line. Consumers are of two types, including average consumers, who value the niche product less than the mass product, and snob consumers, who evaluate the niche product highly. Results demonstrate that the existence of P2P sharing makes consumers' ownership decisions immaterial, which alleviates difficulties of cannibalization between mass and niche product lines and which therefore encourages firms to widen their product variety. Moreover, to address issues of product design, the model is extended to allow the firm to choose the degree of niche‐serving of the second product line endogenously. Results show that P2P sharing deters a firm from designing a niche for the second product line.
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Tang, Han, Li-Hang Chen und Jiří Friml. „Auxin fluctuation and PIN polarization in moss leaf cell reprogramming“. Plant And Cell Physiology, 18.01.2025. https://doi.org/10.1093/pcp/pcaf008.
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Abstract Auxin and its PIN-FORMED (PIN) exporters are essential for tissue repair and regeneration in flowering plants. To gain insight into the evolution of this mechanism, we investigated their roles in leaves excised from Physcomitrium patens, a bryophyte known for its remarkable cell reprogramming capacity. We used various approaches to manipulate auxin levels, including exogenous application, pharmacological manipulations, and auxin biosynthesis mutants. We observed no significant effect on the rate of cell reprogramming. Rather, our analysis of auxin dynamics revealed a decrease in auxin levels upon excision, which was followed by a local increase before the reprogramming process began. Mutant analysis revealed that PpPINs are required for effective cell reprogramming, and endogenously expressed PpPINA-GFP accumulates polarly at sites that will develop into future filamentous stem cells. In addition, hyperpolarized PpPINA variants carrying mutated phosphorylation sites showed a marked delay in reprogramming, whereas endogenous or non-polar versions do not have this effect. These results underscore that both, the levels and the polarity of PpPINA are important for efficient cell reprogramming. Overall, these findings highlight the pivotal role of PIN polarity in plant regeneration. Furthermore, they suggest that understanding polarity mechanisms could have broader implications for improving regenerative processes across various plant species.
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Caruso, German, Lautaro Chittaro, Maria Emilia Cucagna und Luis Pedro Espana. „From bad to worse: The economic impact of COVID-19 in developing countries. Evidence from Venezuela“. Latin American Economic Review, 18.10.2021, 1–22. http://dx.doi.org/10.47872/laer.v30.38.
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Policy responses to COVID-19 affected the dynamic of eco¬nomic growth and labor markets worldwide, hitting econom¬ically harder on developing countries. These policies involved economic lockdowns that included the shutdown of the main statistical exercises, making it almost impossible to assess the breadth and variety of their effects. Using a phone survey, this paper examines the impact of the quarantine implemented in Venezuela on labor market outcomes. The identification strategy exploits the exogenous variation in the severity of the lockdown in different regions of the country. The main result indicates a 16.5 percentage points reduction in employment, while in regions with severe lockdowns the reduction has been 13.8 p.p. larger. In particular, the self-employed and informal¬ly employed were hard hit by the lockdown. To cope with this effect, households sold their productive assets, reduced their savings, sought for alternative income sources and looked for help from relatives. This paper does not find a differential ef¬fect on the number of COVID-19 cases in more severe lock¬down settings. Results are robust to endogenous migration and alternative specifications.
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Hildreth, Kelsey, Haley Overby, Sean Kodani, Christophe Morisseau, Bruce Hammock, Ahmed Bettaieb und Ling Zhao. „Soluble Epoxide Hydrolase Inhibitor t-AUCB Promotes Murine Brown Adipogenesis: Role of PPAR Gamma and PPAR Alpha (P21-069-19)“. Current Developments in Nutrition 3, Supplement_1 (01.06.2019). http://dx.doi.org/10.1093/cdn/nzz041.p21-069-19.
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Abstract Objectives Brown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. Methods The effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. Results We report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. Conclusions Our findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. Funding Sources The work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).
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Fan, Baoyan, Wanlong Pan, Xinli Wang, Michael Chopp, Zheng Gang Zhang und Xian Shuang Liu. „Abstract TMP25: Long Non-Coding RNA Mediates Stroke-Induced Neurogenesis“. Stroke 51, Suppl_1 (Februar 2020). http://dx.doi.org/10.1161/str.51.suppl_1.tmp25.
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Background and Purpose: Adult neurogenesis contributes to functional recovery after stroke. Long non-coding RNAs (lncRNAs) regulate stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. Methods and Results: Using lncRNA array and in situ hybridization, we analyzed lncRNA profiles of adult neural stem cells (NSCs) isolated from the subventricular zone neurogenic region in rats subjected to middle cerebral artery occlusion. We found that H19 was the most highly upregulated lncRNA (19 fold) in ischemic NSCs compared with non-ischemic NSCs. Reduction of endogenous H19 in NSCs by CRISPR-Cas9 genome editing significantly decreased the proliferation and increased the apoptosis of ischemic NSCs, as assayed by the number of BrdU + cells (56±5% vs 22±3%, p<0.01, n=3) and Caspase-3/7 activity compared to NSCs transfected with scrambled small guide RNA (sgRNA). Knockdown of H19 significantly decreased the number of Tuj1 + neuroblasts (8±2% vs 5±0.4%, p<0.01, n=3) and NG 2 + oliogodendrocyte progenitor cells (10±1% vs 5±0.3%, p<0.01, n=3), suggesting that deletion of H19 suppresses the proliferation and survival and blocks the differentiation of NSCs into neurons and oligodendrocytes. Additional RNA-sequencing and bioinformatics analyses revealed that genes deregulated by H19 knockdown were involved in transcription, apoptosis, proliferation, cell cycle and response to hypoxia. Western blot analysis validated that loss-of-function and gain-of-function of H19 significantly increased and reduced, respectively, the transcription of cell cycle-related genes including p27. Using ChIRP assay, we found that upregulated H19 in NSCs was physically associated with EZH2 which catalyzes the repressive H3K27me3 histone marker. Knockdown of H19 significantly reduced the enrichment of H3K27me3 at the promoter of p27, leading to the upregulation of p27 expression and consequently inhibition of NSC proliferation. Conclusions: H19 mediates stroke-induced neurogenesis by regulating genes involved in cell cycle and survival through the interaction with chromatin remodeling proteins. Our data provide novel insights into epigenetic regulation of gene expression by lncRNA in neurogenesis.
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Wang, Jingyu, Lintao Xu, Deqing Peng, Yongjian Zhu, Zhaowen Gu, Ying Yao, Heyangzi Li et al. „IFN-γ-STAT1-mediated CD8+ T-cell-neural stem cell cross talk controls astrogliogenesis after spinal cord injury“. Inflammation and Regeneration 43, Nr. 1 (13.02.2023). http://dx.doi.org/10.1186/s41232-023-00263-9.
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Abstract Background Spinal cord injury (SCI) causes nearly all patients to suffer from protracted disabilities. An emerging therapeutic strategy involving the recruitment of endogenous neural stem cells (NSCs) has been developed. However, endogenous NSCs in the adult spinal cord differentiate into mostly astrocytes after traumatic injury, forming glial scars, which is a major cause of regeneration failure in SCI. Thus, understanding which factors drive the activation and differentiation of endogenous NSCs after SCI is critical for developing therapeutic drugs. Methods The infiltration, state, and location of CD8+ T cells in spinal cord after traumatic injury were analyzed by flow cytometry and immunofluorescence (IF) staining. The Basso Mouse Scale (BMS) scores and rotarod testing were used for motor behavioral analysis. NSCs were co-cultured with CD8+ T cells. EdU assay was used to detect proliferative cells. Western blotting was used to analyze the expression levels of STAT1, p-STAT1, and p27. ChIP-seq and ChIP-qRT-PCR analyses were used to detect the downstream of STAT1. Nestin-CreERT2::Ai9 transgenic mice were used to genetic lineage tracing of Nestin+ NSCs after SCI in vivo. Results A prolonged increase of activated CD8+ T cells occurs in the injured spinal cords. The behavioral analysis demonstrated that the administration of an anti-CD8 antibody promotes the recovery of locomotor function. Then, we discovered that CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1 pathway in vitro. ChIP-seq and ChIP-qRT-PCR analysis revealed that STAT1 could directly bind to the promoters of astrocyte marker genes GFAP and Aldh1l1. Genetic lineage tracing of Nestin+ NSCs demonstrated that most NSCs differentiated into astrocytes following SCI. Depleting CD8+ T cells reduced the differentiation of NSCs into astrocytes and instead promoted the differentiation of NSCs into oligodendrocytes. Conclusion In conclusion, CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1-GFAP/Aldhl1l axis. Our study identifies INF-γ as a critical mediator of CD8+ T-cell-NSC cross talk and a potential node for therapeutic intervention in SCI.
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Rodgers, Krista M., Jared T. Ahrendsen, Frank A. Strnad, Joan C. Yonchek, Wendy B. Macklin, Richard J. Traystman und Paco S. Herson. „Abstract 70: Neurogenesis in the Pediatric Brain Following Ischemic Stroke: a Potential Target for Endogenous Regeneration and Repair“. Stroke 47, suppl_1 (Februar 2016). http://dx.doi.org/10.1161/str.47.suppl_1.70.
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Introduction: Following stroke, neurons are seriously damaged or die, impairing local brain function and contributing to long-term disability. Mounting evidence suggests that stroke recovery in children is enhanced compared to adults. Neurogenesis, a process involving the generation of functionally integrated neurons from progenitor cells, may play a role in enhanced plasticity and neuronal repair. Stroke-induced neurogenesis in adults involves massive proliferation and migration of newborn neurons, however these newborn neurons go on to die, never repopulating areas of damage. We tested the hypothesis that neurogenesis in the young brain effectively repopulates injured regions following ischemia. Methods: Stroke was induced in adult (2-3 mo, n=21) and pediatric (P20-25, n=21) mice by 45-min right middle cerebral artery occlusion (MCAo). Bromodeoxyuridine (BrdU) was injected on days 3 and 4, and mice sacrificed at 24 hr, 7 d or 30 d after recovery from MCAo. Immunohistochemistry was performed to assess cellular proliferation and neurogenesis. Results: The results revealed extensive neuronal cell death in the striatum of both pediatric and adult mice at 24 hr and 7 d after stroke. Remarkably, significant numbers of healthy, mature neurons (NeuN+) were observed in the striatum of pediatric mice at 30 d post-injury. Birth-dating with BrdU demonstrated robust, ischemia-induced proliferation of neural progenitor cells in both adult and pediatric brain. Consistent with previous reports, we observed very few mature NeuN+ neurons double labeled with BrdU in the injured adult brain. In contrast, significant numbers of BrdU+NeuN cells were observed in the pediatric brain 30 d after MCAo, indicative of mature neurons and most importantly, with COUP-TF1-interacting protein 2 (Ctip2) expression, a marker of medium spiny striatal neurons. Conclusion: Our results indicate that cerebral ischemia in pediatric mice increases neurogenesis and migration to sites of damage, and supports the possibility of true neuronal replacement in the pediatric brain. These findings have exciting implications for heightened restorative processes in the pediatric brain microenvironment.
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Nurhayati, Retno, Yoshihiro Ojima, Naoki Nomura und Masahito Taya. „Promoted megakaryocytic differentiation of K562 cells through oxidative stress caused by near ultraviolet irradiation“. Cellular and Molecular Biology Letters 19, Nr. 4 (01.01.2014). http://dx.doi.org/10.2478/s11658-014-0215-3.
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AbstractReactive oxygen species (ROS) have been proven to be important activators for various cellular activities, including cell differentiation. Several reports showed the necessity of ROS during cell differentiation of the megakaryocytic (MK) lineage. In this study, we employed near ultraviolet (near-UV) irradiation to generate endogenous oxidative stress in an MK differentiation process of K562 cells with phorbol 12-myristate 13-acetate (PMA) induction. A significant increase in the intracellular ROS level was detected on day 1 after near-UV irradiation. In the initial stage of differentiation, a shifted fraction of G1 and G2 phase cells was obtained using near-UV irradiation, giving an increased percentage of G2 phase cells (up from 31.1 to 68.7%). The near-UV irradiation-induced upregulation of the p21 gene, which is a cell cycle inhibitor, suggested that the G2 phase cells were prevented from undergoing cell division. It was found that the percentage of high ploidy (8N and 16N) cells was enhanced significantly at the later stage of the K562 cell culture with near-UV irradiation. Moreover, time-lapse analysis showed that near-UV irradiation encouraged the expression of CD41, a specific surface marker of megakaryocytes. This is the first report that the elevated oxidative stress through the near-UV irradiation promoted the MK differentiation of PMA-induced K562 cells.
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Suzuki, Nobuharu, Kaori Sekimoto, Chikako Hayashi, Yo Mabuchi, Eriko Grace Suto, Tetsuya Nakamura und Chihiro Akazawa. „A Novel Murine Experimental System for Analyzing Differentiation of Oligodendrocyte Precursor Cells Using Sox10‐Venus Mice“. FASEB Journal 30, S1 (April 2016). http://dx.doi.org/10.1096/fasebj.30.1_supplement.869.10.
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In the central nervous system (CNS), myelin surrounding axons are formed by oligodendrocytes. Dysfunction of oligodendrocytes in CNS myelination results in neurological disorders, such as leukodystrophy and multiple sclerosis. However, the detailed mechanism of oligodendrocyte differentiation, including cellular process formation, and of myelination has not been elucidated yet, particularly since purification of oligodendrocyte precursor cells (OPCs) from mice, but not rats, is not efficient. We recently created and established a transgenic mouse line that expresses a fluorescent protein Venus driven by the promoter of the Sox10 gene. Endogenous Sox10 is specifically expressed in OPCs and oligodendrocytes in the CNS. In the present study, we have characterized Venus‐positive (+) cells from the brain of Sox10‐Venus mice for the analysis of oligodendrocyte differentiation. First, we purified Venus (+) cells from the brains at postnatal day 0–2, when OPCs have not differentiated into oligodendrocytes, by flow cytometry. Most Venus (+) cells expressed an OPC marker NG2, but not other glial cell markers including galactocerebroside (GalC), a marker for oligodendrocytes. After induction of differentiation with the serum‐free differentiation medium, an increase of GalC‐positive oligodendrocytes and decrease of NG2‐positive OPCs were observed in the Venus (+) culture. These results suggest that OPCs were efficiently purified and differentiated into oligodendrocytes using the Sox10‐Venus system. In addition, a time‐lapse analysis of the Venus (+) OPC differentiation was performed and showed that Venus (+) OPCs dynamically changed their cell morphology with highly branched cellular processes during their differentiation into oligodendrocytes. We further found that Venus (+) OPCs were differentiated into type II astrocytes with the serum‐containing differentiation medium. In vivo, Venus (+) cells expressed NG2 at P0 and both NG2 and myelin basic protein, an oligodendrocyte marker, at P20, when numerous oligodendrocytes are differentiated from OPCs and form myelin. Moreover, some of S‐100‐positive astrocytes expressed Venus in the ventral cortex. Together, the Sox10‐Venus mouse system is useful for analyzing differentiation and multipotency of murine OPCs.
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Preishuber-Pflügl, Julia, Daniela Mayr, Veronika Altinger, Susanne M. Brunner, Andreas Koller, Christian Runge, Anja-Maria Ladek et al. „Pericyte-derived cells participate in optic nerve scar formation“. Frontiers in Physiology 14 (18.04.2023). http://dx.doi.org/10.3389/fphys.2023.1151495.
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Introduction: Pericytes (PCs) are specialized cells located abluminal of endothelial cells on capillaries, fulfilling numerous important functions. Their potential involvement in wound healing and scar formation is achieving increasing attention since years. Thus, many studies investigated the participation of PCs following brain and spinal cord (SC) injury, however, lacking in-depth analysis of lesioned optic nerve (ON) tissue. Further, due to the lack of a unique PC marker and uniform definition of PCs, contradicting results are published.Methods: In the present study the inducible PDGFRβ-P2A-CreERT2-tdTomato lineage tracing reporter mouse was used to investigate the participation and trans-differentiation of endogenous PC-derived cells in an ON crush (ONC) injury model, analyzing five different post lesion time points up to 8 weeks post lesion.Results: PC-specific labeling of the reporter was evaluated and confirmed in the unlesioned ON of the reporter mouse. After ONC, we detected PC-derived tdTomato+ cells in the lesion, whereof the majority is not associated with vascular structures. The number of PC-derived tdTomato+ cells within the lesion increased over time, accounting for 60–90% of all PDGFRβ+ cells in the lesion. The presence of PDGFRβ+tdTomato- cells in the ON scar suggests the existence of fibrotic cell subpopulations of different origins.Discussion: Our results clearly demonstrate the presence of non-vascular associated tdTomato+ cells in the lesion core, indicating the participation of PC-derived cells in fibrotic scar formation following ONC. Thus, these PC-derived cells represent promising target cells for therapeutic treatment strategies to modulate fibrotic scar formation to improve axonal regeneration.
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Campbell, Ross, Marie-Helena Docherty, David Baird, David Ferenbach und Katie Mylonas. „#6679 ANALYSIS OF MACROPHAGE POPULATIONS IN THE KIDNEY WITH RENAL TUBULAR CELL-SPECIFIC SENESCENCE INDUCTION“. Nephrology Dialysis Transplantation 38, Supplement_1 (Juni 2023). http://dx.doi.org/10.1093/ndt/gfad063c_6679.
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Abstract Background and Aims Ageing is a risk factor for multiple diseases, including kidney disease. To improve quality of life, pharmaceutical treatments are given to ageing patients, often with side-effects and cross-reactions. Research is needed into the effects and mechanisms of ageing in order to identify new druggable targets to improve quality of life. Cells can become senescent with age and are seen at sites of tissue injury. Senescent cells (SCs) undergo irreversible cell-cycle arrest, but remain metabolically active, producing a range of signalling molecules called the senescence associated secretory phenotype (SASP, made up of pro-inflammatory/fibrotic elements). Senescent cells within the kidney post-acute kidney injury (AKI) may be involved in ongoing damage, leading in some cases to chronic kidney disease (CKD), possibly due to interactions with macrophages. We hypothesise that the recruitment of monocytes/macrophages by senescent cells is due to the signalling components present in their SASP. Method Pax8 is renal tubule marker. We use Pax8-creERT2;mdm2 fl/fl mice to study kidney-specific senescence induction in absence of age/injury. Upon administration of tamoxifen, the mdm2 alleles are floxxed out by cre recombinase, allowing p53 to stabilise and activate the p21CIP1 cell-cycle inhibitor, inducing senescence in Pax8+ kidney-specific cells. The use of an endogenous tdTomato reporter allows visualisation of cells undergoing successful recombination upon the administration of tamoxifen (TAM). Results As expected, cell-cycle inhibitor/SC marker, Cdkn1a was upregulated by qPCR in TAM-treated young murine whole kidneys 10-fold compared to controls (p = 0.0173) (Figure 1). Ccl2 (monocyte chemoattractant) was up-regulated 20-fold in young (p = 0.0405) TAM-treated TG mice vs controls (Figure 1). In agreement with an increase of Ccl2 transcripts, there was a significant increase of renal macrophages as quantified by flow cytometry of kidney digests (p = 0.0141) in TAM treated young TG mice. Immunofluorescence staining of transgenic mice revealed an increase of Iba1 positive macrophages, correlated with staining of tdTomato tubules, suggesting that increase in macrophage numbers was in response to senescence induction (Figure 2). Iba1-p21 co-stains show a increases in p21 positive cells in TAM-treated transgenic mice, evidently a result of construct activation and appear in proximity to the macrophages (Figure 2). Conclusion Apparent increases in macrophages due to the presence of senescent cells may indicate one of the mechanisms by which AKI progresses to CKD, particularly if inflammatory macrophage phenotypes are present and persist. Macrophage and monocyte populations fluctuate with age and the use of this model allows analysis of macrophage recruitment/polarisation due to senescent cell burden in the absence of age/injury and possible confounding factors, in young mice.
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Crews, Fulton T., Rachael Fisher, Chloe Deason und Ryan P. Vetreno. „Loss of Basal Forebrain Cholinergic Neurons Following Adolescent Binge Ethanol Exposure: Recovery With the Cholinesterase Inhibitor Galantamine“. Frontiers in Behavioral Neuroscience 15 (26.02.2021). http://dx.doi.org/10.3389/fnbeh.2021.652494.
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Binge drinking and alcohol abuse are common during adolescence and cause both cognitive deficits and lasting cholinergic pathology in the adult basal forebrain. Acetylcholine is anti-inflammatory and studies using the preclinical adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2 day on/2 day off from postnatal day [P]25 to P54) model of human adolescent binge drinking report decreased basal forebrain cholinergic neurons (BFCNs) and induction of proinflammatory genes that persist long into adulthood. Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying mechanisms contributing to the persistent loss of BFCNs are unknown. We report that treatment with the cholinesterase inhibitor galantamine (4.0 mg/kg, i.p.) administered during AIE (i.e., P25–P54) or following the conclusion of AIE (i.e., P57–P72) recovered the persistent loss of cholinergic neuron phenotype markers (i.e., ChAT, TrkA, and p75NTR) and somal shrinkage of residual ChAT + neurons known to persist in AIE-exposed adults. Galantamine treatment also recovered the AIE-increased expression of the proinflammatory receptors TLR4 and RAGE, the endogenous TLR4/RAGE agonist HMGB1, and the transcription activation marker pNF-κB p65. Interestingly, we find BFCNs express TLR4 and RAGE, and that AIE treatment increased pNF-κB p65 expression in adult ChAT + IR neurons, consistent with intracellular HMGB1-TLR4/RAGE signaling within BFCNs. AIE increased epigenetic transcription silencing markers (i.e., H3K9me2 and H3K9me3) in the adult basal forebrain and H3K9me2 occupancy at cholinergic phenotype gene promoters (i.e., ChAT and TrkA). The finding of no AIE-induced changes in total basal forebrain NeuN + neurons with galantamine reversal of AIE-induced ChAT + neuron loss, TLR4/RAGE-pNF-κB p65 signals, and epigenetic transcription silencing markers suggests that AIE does not cause cell death, but rather the loss of the cholinergic phenotype. Together, these data suggest that AIE induces HMGB1-TLR4/RAGE-pNF-κB p65 signals, causing the loss of cholinergic phenotype (i.e., ChAT, TrkA, and p75NTR) through epigenetic histone transcription silencing that result in the loss of the BFCN phenotype that can be prevented and restored by galantamine.
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Simko, Stephanie, Jacob Johnson, Guillermo Flores, Christopher C. Barney und Leah Chase. „Exposure to Homocysteic Acid Early in Postnatal Development Leads to a Mixed Depressive/Manic Behavioral Phenotype and Changes in NMDA Receptor Expression“. FASEB Journal 31, S1 (April 2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.1076.3.
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Homocysteic acid (HCA), a NMDA receptor agonist, is an endogenous metabolite formed from the oxidation of homocysteine. Since hyperhomocysteinemia is a risk factor for several neuropsychiatric disorders, including bipolar disorder and major depressive disorder (MDD), we tested the hypothesis that elevated HCA levels in developing rats may induce alterations in NMDA receptor expression and the development of behaviors associated with MDD and/or bipolar disorder. Twenty postnatal male and twenty female rats were injected daily with either HCA or saline from day P3 to P21. HCA‐treated rats displayed increased risk‐taking behavior, reduced social behavior, novelty‐induced hyper‐locomotion, anhedonia in the saccharine preference test, and reduced spatial learning in the Morris water maze, consistent with a depressive state with manic tendencies. Interestingly, female rats were more sensitive to the effects of HCA than male rats. As expected, HCA treatment had no effect on motor coordination (Rotarod) or startle behavior (paired‐pulse inhibition). In addition to these behavioral changes, we observed that HCA led to an increase in expression of the GABAergic marker, GAD‐67, in the cortex, but not the hippocampus, of both male and female rats. We also observed a significant change in the NMDAR2b:NMDAR2a subunit expression ratio in the cortex. Specifically, HCA triggers an increase in the NMDAR2b:NMDAR2a ratio in male rats, while female rats exhibit a decrease in this ratio. We also observed that HCA triggered an increase in NMDAR2a expression in the hippocampus in both males and females. Finally, HCA also led to a decrease in NMDAR2b expression in females, but an increase in NMDAR2b expression males in the hippocampus. We are currently examining the effect of HCA exposure on NMDAR1 subunit expression. Collectively, these data suggest that early postnatal exposure to HCA may lead to a mixed manic/depressive phenotype that may also be accompanied by changes in GABAergic signaling in the cortex. Given the role that NMDA receptors are proposed to play in regulating GABAergic interneuron activity and the regulation of mood, we suggest that this may serve as a novel animal model for studies of complex mood disorders such as bipolar disorder or major depressive disorder.Support or Funding InformationThis research was supported by the Hope College Neuroscience Program, Biology Department and Chemistry Department.