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Fu, Xudong, Mohamed Nadhir Djekidel, and Yi Zhang. "A transcriptional roadmap for 2C-like–to–pluripotent state transition." Science Advances 6, no. 22 (May 2020): eaay5181. http://dx.doi.org/10.1126/sciadv.aay5181.
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In mouse embryonic stem cell (ESC), a small cell population displays totipotent features by expressing a set of genes that are transiently active in 2-cell–stage embryos. These 2-cell–like (2C-like) cells spontaneously transit back into the pluripotent state. We previously dissected the transcriptional dynamics of the transition from pluripotency to the totipotent 2C-like state and identified factors that modulate the process. However, how 2C-like cells transit back into the pluripotent state remains largely unknown. In this study, we analyzed the transcriptional dynamics from the 2C-like state to pluripotent ESCs and identified an intermediate state. The intermediate state characterized by two-wave step up-regulation of pluripotent genes is different from the one observed during the 2C-like entry transition. Nonsense-mediated Dux mRNA decay plays an important role in the 2C-like state exit. Thus, our study not only provides a transcriptional roadmap for 2C-like–to–pluripotent state transition but also reveals a key molecular event driving the transition.
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Xie, Sheila Q., Bryony J. Leeke, Chad Whilding, Ryan T. Wagner, Ferran Garcia-Llagostera, YiXuan Low, Paul Chammas, et al. "Nucleolar-based Dux repression is essential for embryonic two-cell stage exit." Genes & Development 36, no. 5-6 (March 1, 2022): 331–47. http://dx.doi.org/10.1101/gad.349172.121.
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Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.
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Wu, Kaixin, He Liu, Yaofeng Wang, Jiangping He, Shuyang Xu, Yaping Chen, Junqi Kuang, et al. "SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States." Cell Reports 30, no. 1 (January 2020): 25–36. http://dx.doi.org/10.1016/j.celrep.2019.12.010.
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Srinivasan, Rajini, Nataliya Nady, Neha Arora, Laura J. Hsieh, Tomek Swigut, Geeta J. Narlikar, Mark Wossidlo, and Joanna Wysocka. "Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage." Science Advances 6, no. 12 (March 2020): eaaz9115. http://dx.doi.org/10.1126/sciadv.aaz9115.
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Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence–dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.
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Deng, Yilun, Justin Drerup, Xinyue Zhang, Ryan Reyes, Jenny Mendez, Aravind Kancharla, Myrna Garcia, et al. "690 CD122-selective IL-2 complexes treat ovarian carcinomas, induce Treg fragility and promote T cell stem cells." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A729—A731. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0690.
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BackgroundOvarian cancer (OC) responds poorly to immunotherapies. Regulatory T cells (Treg) engage IL-2 by high-affinity CD25 for differentiation and function,1 and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122 (Teff) over CD25 (Tregs).MethodsOrthotopic ID8agg-luc mouse OC burden was measured by in vivo imaging. Tumor, ascites and draining lymph nodes (TDLN) were analyzed by flow and tSNE. IL-2c was complexed using 1.5 µg/mouse IL-2 and 7.5 µg/mouse aIL-2 (clone JES6-5H4) before i.p. injection every other day x 4 starting at day 7. antiPD-L1 was given at 100ug/mouse every 3 days x 4 starting from Day 11. FIR mice2 were used to sort live Tregs.ResultsIL-2c but not antiPD-L1 potently inhibits ID8agg (figure 1). IL-2c decreased ascites Treg functional markers (e.g., CD25, granzymeB) while upregulating the same markers on Teffs (figure 2). IL-2c inhibited Treg suppression in ascites while TDLN Tregs were unaffected (figure 3). tSNE showed great similarity of TDLN Tregs treated with isotype and IL-2c while ascites Tregs after IL-2c showed a fragile phenotype (e.g., increased PD-1, T-bet, and IFNgamma with maintained FoxP3 expression [figure 4]) which is known to contribute to better response to cancer immunotherapy.3 4 We observed a complete reduction of tumor bioluminescence with IL-2c and antiPD-L1 combo treatment in nearly all subjects significantly exceeding effect of IL-2c alone (figure 5). A CD8+CXCR5+TCF-1+ T cell stem cell (TCSC) population reportedly improves immune checkpoint blockade efficacy.5 6 Since CD122 is regulated by TCF-1,7 we explored the effect of IL-2c on these TCSC. IL-2c significantly induced a CD8+TCF-1+ TCSC population in ID8agg tumors (figure 6), possibly through a positive feedback loop by further enhancing CD122 expression on TCF-1+, but not TCF-1- cells (figure 7). tSNE analysis of detailed immune phenotype of IL-2c induced TCSC revealed that these TCSC differed from those induced by antiPD-L1. In ID8agg, antiPD-L1-induced TCSC are mostly CXCR5+ and PD1+, consistent with previous reports in other cancers3 4 while IL-2c-induced TCSC were PD1- (figure 8), expressed CCR2 and CXCR3, and produced TNFalpha (figure 9).Abstract 690 Figure 1IL-2c but not aPD-L1 treats ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, or IL-2c measured by in vivo imaging. Arrows indicate treatments.Abstract 690 Figure 2IL-2c inhibits functional markers on tregs and promotes teffExpression of CD25 and granzymeB were measured by flow cytometry in indicated population from isotype or IL-2c treated ascites 3 weeks after final IL-2c dose.Abstract 690 Figure 3IL-2c reduces ascites Treg suppressive functionTregs sorted from ascites or TDLN of isotype or IL-2c treated FIR mice co-cultured with Teff cells. Percent suppression of Teff cells proliferation shown.Abstract 690 Figure 4IL-2c induces Treg fragility in ascites but not TDLN tSNE analysis on CD45+CD3+CD4+FoxP3+ cells from ascites and TDLN of isotype and IL-2c treated ID8agg-luc challenged mice. Right, representative bar graphs of flow data.Abstract 690 Figure 5IL-2c and aPD-L1 combo treatment effectively cures ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, IL-2c or combo measured by in vivo imaging. Arrows indicate treatments.Abstract 690 Figure 6IL-2c induces a CD8+TCF-1+ population in ID8aggID8agg-luc tumors analyzed by flow cytometry gated on CD45+CD3+CD8+ cells.Abstract 690 Figure 7IL-2c induces CD122 on CD8+TCF-1+ but not TCF-1- cellsCD122 expression was measured by flow cytometry in CD8+TCF-1+ and CD8+ TCF-1- cells from tumors treated with isotype or IL-2c.Abstract 690 Figure 8CD8+TCF1+ cells from ID8agg analyzed by flow cytometry plus tSNE. Cells expressing CXCR5 (blue) and PD1 (red) indicated in overlayAbstract 690 Figure 9CD8+TCF1+ cells from ID8agg analyzed by flow cytometryConclusionsWe define two novel IL-2c effects: inducing Treg fragility therefore reducing immunosuppression while promoting TCSC that could enhance effective anti-tumor immunity. Current work tests if effects are related and help efficacy, and mechanisms for IL-2c Treg effects. We also show that elicited TCSC differ by treatment and tumor, requiring additional investigations.AcknowledgementsThis work is supported by CPRIT Research Training Award (RP 170345), Ovarian Cancer Research Alliance Ann and Sol Schreiber Mentored Investigator Award to YD and R01 CA205965to TC.Ethics ApprovalAll mice studies were approved by UT Health San Antonio Institutional Animal Care and Use Committee (IACUC). Approval number 20150093AR, 20140001AR, 20170035AR, 20140039AR, 20140027AR, 20090128AR, 20120071AR, 20180021AR.ReferencesMalek TR: The biology of interleukin-2. Annu Rev Immunol 2008, 26:453–479.Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C: In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc 2007, 2(7):1789–1794.Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, et al: Interferon-gamma Drives Treg Fragility to Promote Anti-tumor Immunity. Cell 2017, 169(6):1130–1141e1111.Overacre-Delgoffe AE, Vignali DAA: Treg Fragility: A Prerequisite for Effective Antitumor Immunity? Cancer Immunol Res 2018, 6(8):882–887.Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak J, Mavilio D, Alloisio M, Ferrari F, Lopci E, et al: High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors. J Exp Med 2018.Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, et al: Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018, 175(4):998–1013 e1020.Jeevan-Raj B, Gehrig J, Charmoy M, Chennupati V, Grandclement C, Angelino P, Delorenzi M, Held W: The Transcription Factor Tcf1 Contributes to Normal NK Cell Development and Function by Limiting the Expression of Granzymes. Cell Rep 2017, 20(3):613–626.
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Hu, Zhenhua, Dennis Eng Kiat Tan, Gloryn Chia, Haihan Tan, Hwei Fen Leong, Benjamin Jieming Chen, Mei Sheng Lau, et al. "Maternal factor NELFA drives a 2C-like state in mouse embryonic stem cells." Nature Cell Biology 22, no. 2 (January 13, 2020): 175–86. http://dx.doi.org/10.1038/s41556-019-0453-8.
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FLORES-CRUZ, J. A., G. E. DELGADO, J. E. CONTRERAS, M. QUINTERO, L. NIEVES, and P. GRIMA-GALLARDO. "STRUCTURAL CHARACTERIZATION OF THE NEW DIAMOND-LIKE SEMICONDUCTOR CuNbGaSe3." Periódico Tchê Química 15, no. 29 (January 20, 2018): 228–33. http://dx.doi.org/10.52571/ptq.v15.n29.2018.228_periodico29_pgs_228_233.pdf.
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The chalcogenide compound CuNbGaSe3, belonging to the system I-II-III-VI3, has been investigated by means of X-ray powder diffraction and its crystal structure has been refined by the Rietveld method.This is a material of the semiconductor type, which improves the properties of a simple semiconductor like CuGaSe2 because it ads spintronic applications due to its magnetic behavior. The powder pattern was composed by 94.2% of the principal phase CuNbGaSe3 and 5.8% of the secondary phase Cu0.667NbSe2. This material crystallizes with a CuFeInSe3-type structure in the tetragonal space group P4 2c (Nº 112), unit cell parameters a = 5.6199(4) Å, c = 11.0275(2) Å, V = 348.28(4) Å3, with a normal adamantane-structure where occurs a degradation of symmetry from the chalcopyrite structure I4 2d to a related structure P4 2c.
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Gómez-Redondo, Isabel, Priscila Ramos-Ibeas, Eva Pericuesta, Raúl Fernández-González, Ricardo Laguna-Barraza, and Alfonso Gutiérrez-Adán. "Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion." International Journal of Molecular Sciences 21, no. 11 (June 9, 2020): 4115. http://dx.doi.org/10.3390/ijms21114115.
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Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.
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Lucci, Valeria, Elena De Marino, Daniela Tagliaferri, Stefano Amente, Alessandra Pollice, Viola Calabrò, Maria Vivo, Geppino Falco, and Tiziana Angrisano. "Identification of Cdk8 and Cdkn2d as new Prame-Target Genes in 2C-Like Embryonic Stem Cells." Genes 13, no. 10 (September 27, 2022): 1745. http://dx.doi.org/10.3390/genes13101745.
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Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame’s downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes, Cdk8 and Cdkn2d, bound by Prame. These two targets, involved in cancer dedifferentiation and pluripotency, have been further validated in RA-resistant ESCs. Here, we observed for the first time that Prame controls the Cdk8 and Cdkn2d genes in ESCs after RA treatment, shedding light on the regulatory network behind the establishment of naïve pluripotency.
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Kime, Cody, Hiroshi Kiyonari, Satoshi Ohtsuka, Eiko Kohbayashi, Michio Asahi, Shinya Yamanaka, Masayo Takahashi, and Kiichiro Tomoda. "Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells." Stem Cell Reports 13, no. 3 (September 2019): 485–98. http://dx.doi.org/10.1016/j.stemcr.2019.07.011.
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Yang, Fan, Xin Huang, Ruge Zang, Jiayu Chen, Miguel Fidalgo, Carlos Sanchez-Priego, Jihong Yang, et al. "DUX-miR-344-ZMYM2-Mediated Activation of MERVL LTRs Induces a Totipotent 2C-like State." Cell Stem Cell 26, no. 2 (February 2020): 234–50. http://dx.doi.org/10.1016/j.stem.2020.01.004.
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Sclavons, Coralie, Sébastien Boutry, Sophie Laurent, Luce Vander Elst, and Robert N. Muller. "Targeting of cell death and neuroinflammation with peptide-linked iron oxide nanoparticles and Gd-DTPA in a mouse model of Parkinson's disease." Journal of Biomedical Engineering and Informatics 2, no. 1 (September 18, 2015): 13. http://dx.doi.org/10.5430/jbei.v2n1p13.
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Parkinson’s disease (PD) is one of the most common neurodegenerative disease and remains difficult to diagnose by conventional methods of early detection. It is characterized by the apoptotic loss of dopaminergic neurons (DN) and a neuroinflammation mainly located in the ventral midbrain (VM). The aim of this work is to study new vectorized contrast agents for magnetic resonance imaging (MRI) detection of PD injured areas by targeting apoptosis and inflammation. Two peptides selected by phage display were used for the experiments: R826 peptide, selected for its affinity for the phosphatidylserine (PS) exposed at the external surface of apoptotic cells, and 2C peptide, selected for its affinity for TNF-α (tumor necrosis factor alpha), one of the most abundant cytokines secreted during inflammation. These peptides were grafted to pegylated iron oxide nanoparticles (PEG-USPIO) and gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) for in vitro and in vivo studies, respectively. PD was simulated on mice with the MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) neurotoxin and its active metabolite MPP+ (1-methyl-4-phenylpyridinium) for in vitro studies. The results showed that PEG-USPIO-R826 and PEG-USPIO-2C enabled the detection of apoptosis and inflammation, respectively, on MPP+-treated culture cells. PEG-USPIO-2C also allowed detection of inflammation on histological brain sections of MPTP-treated mice. The 2C peptide grafted to Gd-DTPA showed encouraging results in MRI detection of injured brain areas in MPTP-treated mice. These observations suggest that a targeting of damaged cells and injured areas by these new specific contrast agents occurs, offering a new tool for early diagnosis of neurodegenerative disorders like PD by MRI.
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Robinson, M. K., W. H. van Zyl, E. M. Phizicky, and J. R. Broach. "TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation." Molecular and Cellular Biology 14, no. 6 (June 1994): 3634–45. http://dx.doi.org/10.1128/mcb.14.6.3634-3645.1994.
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The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.
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Robinson, M. K., W. H. van Zyl, E. M. Phizicky, and J. R. Broach. "TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation." Molecular and Cellular Biology 14, no. 6 (June 1994): 3634–45. http://dx.doi.org/10.1128/mcb.14.6.3634.
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The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.
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Burrack, Kristina Stoermer, Matthew A. Huggins, Geoffrey T. Hart, Aaron J. Johnson, Stephen C. Jameson, and Sara E. Hamilton. "IL-15 complex-stimulated NK cells protect mice from cerebral malaria." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 123.3. http://dx.doi.org/10.4049/jimmunol.198.supp.123.3.
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Abstract Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly 500,000 malaria-related deaths annually. In the experimental mouse model of CM (ECM), mice are inoculated with red blood cells infected with Plasmodium berghei ANKA (PbA) parasites and develop a CM-like disease within 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57Bl/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced ECM. IL-15C treatment stimulates natural killer (NK) and CD8 T cells. Interestingly, adoptive transfer of IL-15C-stimulated NK cells, but not CD8 T cells, prevented ECM. Similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) also induce robust expansion and activation of NK cells, but fail to protect against ECM. Rescue from ECM was not associated with reduced parasitemia at 6 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. Moreover, we found that in vivo IL-15C, but not IL-2C, treatment of mice induces IL-10 expression in NK cells, and that IL-10 is required for IL-15C-mediated protection from ECM. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can also restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.
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Dej, K. J., and A. C. Spradling. "The endocycle controls nurse cell polytene chromosome structure during Drosophila oogenesis." Development 126, no. 2 (January 15, 1999): 293–303. http://dx.doi.org/10.1242/dev.126.2.293.
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Polytene chromosomes exhibit intricate higher order chromatin structure that is easily visualized due to their precisely aligned component strands. However, it remains unclear if the same factors determine chromatin organization in polyploid and diploid cells. We have analyzed one such factor, the cell cycle, by studying changes in Drosophila nurse cell chromosomes throughout the 10 to 12 endocycles of oogenesis. We find that nurse cells undergo three distinct types of endocycle whose parameters are correlated with chromosome behavior. The first four endocycles support complete DNA replication; poorly banded polytene euchromatin progressively condenses during the late S phases to produce blob-like chromosomes. During the unique fifth endocycle, an incomplete late S phase is followed by a mitosis-like state during which the 64C chromosomes dissociate into 32 chromatid pairs held together by unreplicated regions. All the subsequent endocycles lack any late S phase; during these cycles a new polytene chromosome grows from each 2C chromatid pair to generate 32-ploid polytene nuclei. These observations suggest that euchromatin begins to condense during late S phase and that nurse cell polytene chromosome structure is controlled by regulating whether events characteristic of late S and M phase are incorporated or skipped within a given endocycle.
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Nishimura, Hiroyuki, Tasuku Honjo, and Nagahiro Minato. "Facilitation of β Selection and Modification of Positive Selection in the Thymus of Pd-1–Deficient Mice." Journal of Experimental Medicine 191, no. 5 (March 6, 2000): 891–98. http://dx.doi.org/10.1084/jem.191.5.891.
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PD-1 is an immunoglobulin superfamily member bearing an immunoreceptor tyrosine-based inhibitory motif, and disruption of the PD-1 gene results in the development of lupus-like autoimmune diseases. In this study, we examined effects of the PD-1 deficiency on the thymocyte differentiation at the clonal level using T cell receptor (TCR)-β (Vβ8) and TCR-α/β (H-Y and 2C) transgenic mice. In these TCR transgenic lines, PD-1 expression in the thymus was variably augmented, but as in the normal mice, confined largely to the CD4−CD8− thymocytes. The transgenic mice crossed with PD-1−/− mice in the neutral genetic backgrounds exhibited selective increase in the CD4+CD8+ (DP) population with little effect on other thymocytes subsets. Similarly, the absence of PD-1 facilitated expansion of DP thymocytes in recombination activating gene (RAG)-2−/− mice by anti-CD3ε antibody injection. On the other hand, H-Y or 2C transgenic PD-1−/− mice with the positively selecting background showed significantly reduced efficiency for the generation of CD8+ single positive cells bearing the transgenic TCR-α/β in spite of the increased DP population. These results collectively indicate that PD-1 negatively regulates the β selection and modulates the positive selection, and suggest that PD-1 deficiency may lead to the significant alteration of mature T cell repertoire.
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Li, Hongguo, Rui Zhu, Liguo Sun, Yingsen Xue, Zhangying Hao, Zhenghong Xie, Xiangli Fan, and Hongbin Fan. "Effect of Thickness of HA-Coating on Microporous Silk Scaffolds Using Alternate Soaking Technology." BioMed Research International 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/637821.
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Hydroxyapatite (HA) can be coated on various materials surface and has the function of osteogenicity. Microporous silk scaffold has excellent biocompatibility. In this study, alternate soaking technology was used to coat HA on microporous silk scaffolds. However, the cell proliferation was found to decrease with the increasing thickness (cycles of soaking) of HA-coating. This study aims to determine the best thickness (cycles of soaking) of HA-coating on microporous silk scaffolds. The SEM observation showed that group with one cycle of alternate soaking (1C-HA) has the most optimal porosity like non-HA-modified microporous silk scaffolds. The proliferation of osteoblasts has no significant difference between noncoated HA (N-HA) and 1C-HA groups, which are both significantly higher than those in two cycles of soaking (2C-HA) and three cycles of soaking (3C-HA) groups. The transcription levels of specific genes (runx2andosteonectin) in osteoblasts of 1C-HA group were significantly higher than those of N-HA group. Moreover, the levels showed no significant difference among 1C-HA, 2C-HA, and 3C-HA groups. In conclusion, microporous silk scaffold with 1 cycle of HA-coating can combine the biocompatibility of silk and osteogenicity of HA.
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Guo, M., K. O. Chang, M. E. Hardy, Q. Zhang, A. V. Parwani, and L. J. Saif. "Molecular Characterization of a Porcine Enteric Calicivirus Genetically Related to Sapporo-Like Human Caliciviruses." Journal of Virology 73, no. 11 (November 1, 1999): 9625–31. http://dx.doi.org/10.1128/jvi.73.11.9625-9631.1999.
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ABSTRACT Porcine enteric calicivirus (PEC) is associated with diarrhea in pigs, and to date it is the only cultivable enteric calicivirus (tissue culture-adapted [TC] PEC/Cowden). Based on sequence analysis of cDNA clones and reverse transcription-PCR products, TC PEC/Cowden has an RNA genome of 7,320 bp, excluding its 3′ poly(A)+ tail. The genome is organized in two open reading frames (ORFs), similar to the organizations of the human Sapporo-like viruses (SLVs) and the lagoviruses. ORF1 encodes the polyprotein that is fused to and contiguous with the capsid protein. ORF2 at the 3′ end encodes a small basic protein of 164 amino acids. Among caliciviruses, PEC has the highest amino acid sequence identities in the putative RNA polymerase (66%), 2C helicase (49.6%), 3C-like protease (43.7%), and capsid (39%) regions with the SLVs, indicating that PEC is genetically most closely related to the SLVs. The complete RNA genome of wild-type (WT) PEC/Cowden was also sequenced. Sequence comparisons revealed that the WT and TC PEC/Cowden have 100% nucleotide sequence identities in the 5′ terminus, 2C helicase, ORF2, and the 3′ nontranslated region. TC PEC/Cowden has one silent mutation in its protease, two amino acid changes and a silent mutation in its RNA polymerase, and five nucleotide substitutions in its capsid that result in one distant and three clustered amino acid changes and a silent mutation. These substitutions may be associated with adaptation of TC PEC/Cowden to cell culture. The cultivable PEC should be a useful model for studies of the pathogenesis, replication, and possible rescue of uncultivable human enteric caliciviruses.
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Marzotto, Marta, Fabio Arruda-Silva, and Paolo Bellavite. "Fibronectin Gene Up-regulation by Arnica montana in Human Macrophages: Validation by Real-Time Polymerase Chain Reaction Assay." Homeopathy 109, no. 03 (April 20, 2020): 140–45. http://dx.doi.org/10.1055/s-0040-1708044.
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Abstract Background and Aim Arnica montana L. (Arnica m.) is a popular traditional medicine, used for its therapeutic properties in healing traumas, but little is known about its biological action on tissue formation and repair. This new work tested the effects of Arnica m. homeopathic dilutions on human macrophages, key cells in tissue defence and repair. Materials and Methods Macrophages derived from the THP-1 cell line were differentiated with interleukin-4 to induce a ‘wound-healing’-like phenotype, and treated with various dilutions of Arnica m. centesimal (100 times) dilutions (2c, 3c, 5c, 9c, and 15c) or control solvent for 24 hours. RNA samples from cultured cells were analysed by real-time quantitative polymerase chain reaction in five separate experiments. Results Arnica montana at the 2c dilution (final concentration of sesquiterpene lactones in cell culture = 10−8 mol/L) significantly stimulated the expression of three genes which code for regulatory proteins of the extracellular matrix, namely FN1 (fibronectin 1, % increase of 21.8 ± standard error of the mean 4.6), low-density lipoprotein-receptor-related protein 1 (% increase of 33.4 ± 6.1) and heparan sulphate proteoglycan 2 (% increase of 21.6 ± 9.1). Among these genes, the most quantitatively expressed was FN1. In addition, FN1, unlike other candidate genes, was upregulated in cells treated with higher dilutions/dynamisations (3c, 5c, and 15c) of Arnica m. Conclusion The results support evidence that the extracellular matrix is a potential therapeutic target of Arnica m., with positive effects on cell adhesion and migration during tissue development and healing.
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Razzaq, Syeda Saima, Irfan Khan, Nadia Naeem, Asmat Salim, Sumreen Begum, and Kanwal Haneef. "Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells." World Journal of Stem Cells 14, no. 9 (September 26, 2022): 700–713. http://dx.doi.org/10.4252/wjsc.v14.i9.700.
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Takai, A., M. Troschka, G. Mieskes, and A. V. Somlyo. "Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2." Biochemical Journal 262, no. 2 (September 1, 1989): 617–23. http://dx.doi.org/10.1042/bj2620617.
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Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.
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O'Grady, Paul I., Angela Borden, Dominique Vandewiele, Ali Ozgenc, Roger Woodgate, and Christopher W. Lawrence. "Intrinsic Polymerase Activities of UmuD′2C and MucA′2B Are Responsible for Their Different Mutagenic Properties during Bypass of a T-T cis-synCyclobutane Dimer." Journal of Bacteriology 182, no. 8 (April 15, 2000): 2285–91. http://dx.doi.org/10.1128/jb.182.8.2285-2291.2000.
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ABSTRACT In wild-type Escherichia coli, translesion replication is largely dependent upon the UmuD′2C complex (DNA polymerase V [polV]) or its plasmid-encoded homologs, such as MucA′2B. Interestingly, both the efficiency of translesion replication of a T-T cis-syn dimer and the spectra of mutations observed are different in Umu- and Muc-expressing strains. We have investigated whether the polIII core is responsible for these differences by measuring the frequency of dimer bypass, the error rate of bypass, and the resulting mutation spectrum in mutants carrying a deletion of dnaQ (ɛ subunit) or holE (θ subunit) or carrying the dnaQ allele mutD5, which is deficient in proofreading but is competent in the structural function of ɛ, or the dnaE antimutator allelespq-2. The chromosomal copy of the umuDC operon was deleted in each strain, and the UmuDC, UmuD′C, MucAB, or MucA′B proteins were expressed from a low-copy-number plasmid. With only few exceptions, we found that the characteristically different mutation spectra resulting from Umu- and Muc-mediated bypass are maintained in all of the strains investigated, indicating that differences in the activity or structure of the polIII core are not responsible for the observed phenotype. We also demonstrate that the MucA′2B complex is more efficient in promoting translesion replication than the UmuD′2C proteins and show that, contrary to expectation, the T-T dimer is bypassed more accurately by MucA′2B than by UmuD′2C. These results are consistent with the view that in a wild-type cell, the polV-like enzymes are responsible for the spectra of mutations generated during translesion replication and that polIII may simply be required to fix the misincorporations as mutations by completing chromosomal replication. Our observations also show that the mutagenic properties of a lesion can depend strongly on the particular enzyme employed in bypass.
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Wang, Gang Feng, William Nikovits, Mark Schleinitz, and Frank E. Stockdale. "A Positive GATA Element and a Negative Vitamin D Receptor-Like Element Control Atrial Chamber-Specific Expression of a Slow Myosin Heavy-Chain Gene during Cardiac Morphogenesis." Molecular and Cellular Biology 18, no. 10 (October 1, 1998): 6023–34. http://dx.doi.org/10.1128/mcb.18.10.6023.
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ABSTRACT We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif, control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity were found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increased in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form.
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Delorme, Violaine, Xavier Cayla, Grazyna Faure, Alphonse Garcia, and Isabelle Tardieux. "Actin Dynamics Is Controlled by a Casein Kinase II and Phosphatase 2C Interplay on Toxoplasma gondii Toxofilin." Molecular Biology of the Cell 14, no. 5 (May 2003): 1900–1912. http://dx.doi.org/10.1091/mbc.e02-08-0462.
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Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility.
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Zhu, Yezhang, Jiali Yu, Jiahui Gu, Chaoran Xue, Long Zhang, Jiekai Chen, and Li Shen. "Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells." Nucleic Acids Research 49, no. 21 (November 17, 2021): 12167–77. http://dx.doi.org/10.1093/nar/gkab1069.
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Abstract The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.
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Maccioni, R. B., and V. Cambiazo. "Role of microtubule-associated proteins in the control of microtubule assembly." Physiological Reviews 75, no. 4 (October 1, 1995): 835–64. http://dx.doi.org/10.1152/physrev.1995.75.4.835.
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In eukaryotic cells, microtubules, actin, and intermediate filaments interact to form the cytoskeletal network involved in determination of cell architecture, intracellular transport, modulation of surface receptors, mitosis, cell motility, and differentiation. Cytoskeletal organization and dynamics depend on protein self-associations and interactions with regulatory elements such as microtubule-associated proteins (MAPs). The MAP family includes large proteins like MAP-1A, MAP-1B, MAP-1C, MAP-2, and MAP-4 and smaller components like tau and MAP-2C. This review focuses on relevant aspects of MAP function, with emphasis on their roles in modulating cytoskeletal interactions. In this context, MAP expression mechanisms and posttranslational modifications are also discussed. Microtubule-associated proteins have a rather widespread distribution among cells, but certain MAPs have been identified in specific cell types. Within single neurons, MAP-2 is dendritic while tau is preferentially an axonal protein. Their expression is developmentally regulated. Even though MAPs share a capacity to interact with the COOH-terminal tubulin domain, stabilize microtubules, and link them with other cytoskeletal polymers, they exhibit structural differences. However, MAP-2, MAP-4, and tau have common repetitive microtubule-binding motifs. Microtubule-associated proteins not only control cytoskeletal integrity, but they also appear to interact with highly structural elements of cells. Molecular biological approaches permitted localization of new MAPs in cultured mammalian cells and invertebrate organisms and other microtubule-interacting proteins that exhibit transient interactions with microtubules. The structural/functional aspects of several new MAP-like proteins in centrosomes and the mitotic spindle, functionally implicated in cell cycle events, are also analyzed.
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Labasque, Marilyne, Eric Reiter, Carine Becamel, Joël Bockaert, and Philippe Marin. "Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling." Molecular Biology of the Cell 19, no. 11 (November 2008): 4640–50. http://dx.doi.org/10.1091/mbc.e08-04-0422.
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The serotonin (5-hydroxytryptamine; 5-HT)2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca2+-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT2C receptor agonists and their antidepressant-like activity.
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Akiyama, Mitsumasa, Hodaka Sugimoto, Shin-ichiro Inoue, Yohei Takahashi, Maki Hayashi, Yuki Hayashi, Miya Mizutani, et al. "Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase." Plant Physiology 188, no. 4 (December 11, 2021): 2228–40. http://dx.doi.org/10.1093/plphys/kiab571.
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Abstract Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.
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Musarò, Antonio, and Nadia Rosenthal. "Maturation of the Myogenic Program Is Induced by Postmitotic Expression of Insulin-Like Growth Factor I." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 3115–24. http://dx.doi.org/10.1128/mcb.19.4.3115.
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ABSTRACT The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC–IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, β-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC–IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of β1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.
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Chen, Jia, Feng Yu, Ying Liu, Changqing Du, Xiushan Li, Sirui Zhu, Xianchun Wang, et al. "FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis." Proceedings of the National Academy of Sciences 113, no. 37 (August 26, 2016): E5519—E5527. http://dx.doi.org/10.1073/pnas.1608449113.
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Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.
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Poli, Alessandro, Shidqiyyah Abdul-Hamid, Antonio Enrico Zaurito, Francesca Campagnoli, Valeria Bevilacqua, Bhavwanti Sheth, Roberta Fiume, Massimiliano Pagani, Sergio Abrignani, and Nullin Divecha. "PIP4Ks impact on PI3K, FOXP3, and UHRF1 signaling and modulate human regulatory T cell proliferation and immunosuppressive activity." Proceedings of the National Academy of Sciences 118, no. 31 (July 26, 2021): e2010053118. http://dx.doi.org/10.1073/pnas.2010053118.
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Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2. They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.
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Raatz, H., A. Böcking, and S. Hauptmann. "Prognostic Impact of DNA-Image-Cytometry in Neuroendocrine (Carcinoid) Tumours." Analytical Cellular Pathology 26, no. 1-2 (January 1, 2004): 81–88. http://dx.doi.org/10.1155/2004/195478.
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Establishing prognosis proves particularly difficult with neuroendocrine tumours (NETs) as a benign looking histology can be associated with a malignant behaviour. In order to identify prognostic factors we examined 44 gastrointestinal and pulmonary, paraffin‐embedded NETs histologically and immunohistochemically. DNA‐image‐cytometry was used to examine 40 of these. We found that poor differentiation (corresponding to a Soga and Tazawa type D) and infiltrative growth correlated with a poorer prognosis. Moreover, parameters determined by diagnostic DNA cytometry like the 5c‐exceeding rate, the 2c‐deviation index, DNA‐grade of malignancy, DNA‐entropy and the type of DNA histogram were found to be of prognostic relevance. Morphometric parameters like the form factor and the mean nuclear area were relevant for survival, tumour recurrence and metastasis. However, in the multivariate analysis the only independent risk factor was the histological differentiation. The 5c‐exceeding rate is a good objective risk factor, which can be used particularly in cases in which only a fine needle biopsie is available. Direct comparison of the histology and the 5c‐exceeding rate in the multivariate analysis suggests that the 5c‐exceeding rate taken as sole prognostic factor might be of higher prognostic relevance than the histology but larger studies are needed to confirm this.
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Betekhtin, Alexander, Magdalena Rojek, Katarzyna Nowak, Artur Pinski, Anna Milewska-Hendel, Ewa Kurczynska, John Doonan, and Robert Hasterok. "Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture." International Journal of Molecular Sciences 19, no. 12 (November 29, 2018): 3811. http://dx.doi.org/10.3390/ijms19123811.
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Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.
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Guehler, S. R., J. A. Bluestone, and T. A. Barrett. "Immune deviation of 2C transgenic intraepithelial lymphocytes in antigen-bearing hosts." Journal of Experimental Medicine 184, no. 2 (August 1, 1996): 493–503. http://dx.doi.org/10.1084/jem.184.2.493.
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The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.
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Rusconi, Chiara, Antonella Anastasia, Annalisa Chiarenza, Luigi Marcheselli, Federica Cavallo, Sara Rattotti, Barbara Botto, et al. "Outcome of Transformed FL(t-FL) Worsens According to the Timing of Transformation and to the Number of Previous Therapies. A Survey of the Fondazione Italiana Linfomi (FIL)." Blood 126, no. 23 (December 3, 2015): 3933. http://dx.doi.org/10.1182/blood.v126.23.3933.3933.
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Abstract Background: Histologic transformation (HT) refers to a biologic event leading to the development of a high grade non-Hodgkin lymphoma, mostly diffuse large B cell lymphoma (DLBCL), in patients (pts) with an underlying follicular lymphoma (FL), whose recently reported risk is 2% to 3% per year. The prognosis of t-FL has been considered historically very poor, but several studies in the Rituximab (RTX) era suggest that survival may be more favorable than previously recognized. The treatment approach for t-FL is often individualized and pts are generally excluded from clinical trials, so there is a paucity of objective data on the optimal management of t-FL, which still represents an unmet need. The aim of our survey conducted in a large number of pts with histologically confirmed t-FL observed in 9 FIL Centers was to analyze the clinical factors and the different treatment strategies which can predict post-transformation outcome. Methods: One hundred seventy-seven t-FL were retrospectively selected from Institutional datasets; the inclusion time frame was from 2002 to 2014. All Histologic Transformations (HT) were biopsy confirmed. Response assessment was made as recommended by International Workshop criteria. Survival analysis were performed with Kaplan-Meier method and compared by Log-Rank test. Results: T-FL occurred at initial diagnosis (Group 1) in 93 cases (53%): 52 were DLBCL (29%) evolving from a prior FL, 31 (17%) composite lymphoma and 10 (6%) discordant lymphoma. HT occurred after a previous FL diagnosis (Group 2) in 84 pts (47%): 15 pts (8%) were treatment-naïve at HT (Group 2A), 38 pts (21%) transformed at first relapse or progression (Group 2B) and 31 (18%) experienced late HT (Group 2C). Median age at HT was 60 years (range: 20-83). No differences were found between Group 1 and 2 and between Group 2A, 2B and 2C in term of clinical features (age, disease stage, B-symptoms). Group 1 received CHOP/CHOP-like regimens in 75% of pts. RTX was used with chemotherapy (CT) in 92% of pts and in 22% as maintenance. Autologous Stem Cell Transplantation (ASCT) was delivered as consolidation in 14%. Group 2 received CHOP/CHOP-like regimens in 39% of pts, platinum-containing regimens in 14%, high dose sequential therapy in 32%. RTX was added to CT in 71% of cases; 12% received RTX maintenance and 23% ASCT consolidation. CHOP as CT and RTX maintenance were used more often in Group 1 pts. Overall Response Rate (ORR) for Group 1 was 94%, with 77 pts (83%) achieving Complete Response (CR) and 10 (11%) Partial Response (PR).With a median follow-up of 43 months, 5-yr Progression-Free Survival (PFS) and Overall Survival (OS) were 60% and 83%, respectively. ORR for Group 2 was 57%, with 43 pts (51%) obtaining CR and 5 (6%) PR; focusing on the subgroups 2A, 2B, 2C ORR was 80%, 63% and 39%, respectively (p 0.017). The 5-yr OS was 52%, statistically inferior to Group 1 (p <0.001) (Figure 1). The use of RTX with CT and of consolidation ASCT favourably influenced survival only in group 2 but not in group 1 pts in univariate analysis. Survival showed a significant trend (p<0.001) to progressively worsen from Group 1 to Group2c pts (Figure 2). Moreover, considering the number of previous FL treatment lines received by group 2, 5-yr OS was 58% for pts who received 1-2 lines compared to 20% for pts who received > 2 lines (p=0.004) (Figure 3). Conclusion: Outcome of t-FL in the RTX era confirms to be better than reported in historical series and strongly differs among subgroups of pts according to the time of transformation and to the number of pre-HT treatment lines. Pts with FL transformation at lymphoma diagnosis, including composite and discordant cases, or with FL transforming after an initial watch and wait policy, i.e. treatment-naïve, show an excellent outcome with standard immuno-chemotherapy, comparable to that of FL (Federico JCO 2013) and of de novo DLBCL (Cunningham, Lancet Onc 2013). They probably do not benefit from front-line ASCT consolidation. Both ORR and survival are significantly worse in pts with t-FL diagnosed after being treated for FL. However t-FL diagnosed at first relapse/progression in this study obtained a 5-ys OS of 51%, which compares favorably with historical cohorts. On the other hand late transformation has an inferior outcome, which becomes really dismal in the sub-group of pts who transformed after two previous lines for FL, whose median survival is less than one year and clearly represent an unmet clinical need. Disclosures Rusconi: Roche: Honoraria.
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Kasabri, Violet, Shereen Arabiyat, Yusuf Al-Hiari, Hiba Zalloum, Jehad Almaliti, Ahmad Telfah, Yasser K. Bustanji, and Sundus Alalawi. "Fluoroquinolones as a potentially novel class of antidiabesity and antiproliferative compounds: synthesis and docking studies." Canadian Journal of Chemistry 98, no. 10 (October 2020): 635–45. http://dx.doi.org/10.1139/cjc-2020-0162.
Full textAbstract:
Intense efforts by the pharmaceutical industry have been made to identify new targets for obesity diabetes (diabesity). Pancreatic triacylglycerol lipase (PL) inhibition is an interesting putative target for obesity management. Fluoroquinolones (FQs) have been identified as potent inhibitors of PL. The aim of this research was to synthesize novel FQs and evaluate their in vitro antilipolytic and antiproliferative properties. Characterization of the synthesized FQs was carried out with NMR, MS, IR, and EA. Like orlistat, potential FQs’ modulation of PL was quantified colorimetrically (n = 3) and was further supported by docking studies. Compared with cisplatin, FQs’ antiproliferative propensities against a panel of obesity related colorectal cancer cell lines were investigated with Sulforhodamine B assay. Twelve novel FQs (2A–5A, 2B–5B, and 2C–5C) were synthesized and characterized. The PL-IC50 values of tested FQs were in the range of 6.8–165.7 μmol/L. FQ 4A was the most active antiproliferative compound against HCT116 with an IC50 value of 3.5 μmol/L. Their selectivity of growth inhibition for safety examination using normal periodontal ligament fibroblasts (PDL) in comparison with cisplatin’s lack of differential cytotoxicity was reported. Lipophilicity and hydrogen bonding were found essential for both activities. Conclusively, FQs are robustly proven for their emerging in vitro anti-obesity and antiproliferative activities.
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Gollomp, Kandace, Amrita Sarkar, Gowthami Arepally, Douglas B. Cines, M. Anna Kowalska, Lubica Rauova, and Mortimer Poncz. "Understanding How Fc-Modification Transforms a Pathogenic Heparin-Induced Thrombocytopenia (HIT)-like Monoclonal Antibody into a Novel Treatment for Sepsis." Blood 134, Supplement_1 (November 13, 2019): 10. http://dx.doi.org/10.1182/blood-2019-123132.
Full textAbstract:
Sepsis is a dysregulated response to infection that leads to life-threatening organ damage. This process induces the release of neutrophil extracellular traps (NETs), webs of negatively-charged cell-free DNA (cfDNA) complexed with positively-charged histones that capture pathogens but also damage host tissue. We believe that upon presentation, most patients with sepsis have already released a large amount of NETs so that preventing NET release (NETosis), or accelerating their lysis with DNase, may be ineffective or harmful. Murine studies of NET lysis in sepsis have met with mixed results raising the concern that this strategy may lead to the release of captured bacteria and harmful NET degradation products (NDPs) such as cell free (cf) DNA and histones. We propose NET stabilization as an alternative therapy in sepsis. In a microfluidic system, we have shown that NETs bound the platelet chemokine platelet factor 4 (PF4, CXCL4), a highly-positively charged protein that aggregates polyanionic molecules including DNA. As a result of this aggregating effect, PF4 physically compacts NETs without inducing histone release. These compact PF4-NET complexes are resistant to DNase lysis. Using a microfluidic system, in which channels coated with NETs were infused with labeled Staphylococcus aureus particles, we now show that PF4 compaction of NETs markedly enhances their ability to capture bacteria. Likely, PF4, which is known to bind to the bacterial cell wall, cross aggregates pathogens to NET DNA. Thus, without added PF4, little bacteria is entrapped in the microfluidic chamber (Fig. 1A & 1B) and those captured are readily released by the infusion of DNase I (Fig. 1C & 1D). In contrast, when PF4 is added, microbial entrapment is markedly enhanced (Fig. 1A & 1B) and persists even with the infusion of DNase (Fig. 1C & 1D). When human (h) PF4 binds to polyanions it changes conformation, revealing HIT antigenic sites that bind the HIT-like monoclonal antibody KKO. Bound KKO further enhances PF4-NET complex resistance to DNase I in the microfluidic chamber, preventing the release of NDPs and entrapped bacteria upon exposure to DNases. These in vitro observations led us to hypothesize that infused PF4 and/or KKO may serve as a targeted therapeutic in sepsis. However, unmodified KKO stimulates leukocytes and induces a prothrombotic state. Indeed, infused KKO accelerates mortality in murine models of sepsis in mice that express hPF4 (hPF4+) (Fig. 2C). We, therefore, modified KKO using an IgG-specific endoglycosidase to create deglycosylated KKO (DG-KKO) that retains the ability to bind to PF4-NET complexes, but has little capacity to interact with hematopoietic cell Fc receptors or activate complement. DG-KKO binding also protects hPF4-NET complexes from DNase lysis and does not interfere with bacterial capture (Fig. 1B). In a murine lipopolysaccharide (LPS) endotoxemia model, DG-KKO infusion prevented thrombocytopenia, decreased the release of NDPs, and enhanced survival exclusively in hPF4+ mice (not shown). Although treatment with LPS recapitulates many aspects of sepsis, it relies on treatment with a bacterial toxin rather than live pathogens. Therefore, to determine if hPF4 and/or DG-KKO mitigate bacterial-induced sepsis, we repeated our studies using cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Infused hPF4, but more so, infused DG-KKO decreased the severity of thrombocytopenia, reduced NDP release, and improved survival (Fig. 2A-2C). These murine studies demonstrate that PF4 released from activated platelets stabilizes NETs, making them resistant to DNase lysis and enhancing their ability to capture bacteria. Infused PF4 and DG-KKO further enhance NET stability, decrease the release of NDPs, and enhance bacterial entrapment, leading to better outcomes. These studies provide mechanistic insights into how NETs contribute to end-organ damage in sepsis and offer a targeted and novel therapeutic that minimizes their harmful effects, while enhancing their protective properties. Disclosures Arepally: Biokit: Patents & Royalties; Apotex Pharmaceuticals: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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Bellavite, Paolo, Marta Marzotto, Debora Olioso, Mirko Cristofoletti, Maurizio Brizzi, Paola Tononi, Ilaria Pierpaola Dal PrÃÂÂ, and Ubaldo Armato. "Cellular and transcriptional responses of SH-SY5Y human neurocytes following in vitro exposure to Gelsemium sempervirens." International Journal of High Dilution Research - ISSN 1982-6206 11, no. 40 (December 21, 2021): 144–46. http://dx.doi.org/10.51910/ijhdr.v11i40.603.
Full textAbstract:
Background: Gelsemium sempervirens (Gelsemium s.) is a highly toxic plant but is employed at low doses and/or high dilutions as an anxiolytic and antidepressant. Previous investigations in our laboratory [1,2] have shown a significant anxiolytic-like activity of Gelsemium s., using emotional response models in laboratory mice. Although there is some biochemical evidence of a possible role of neurosteroid metabolism [3], the cellular and molecular mechanisms involved in the effects of Gelsemium s. at the level of nervous system are largely unknown. To help determine these pathways, we used human neurocytes (SH-SY5Y cell line) treated in vitro with different dilutions of Gelsemium s. and evaluated their vitality and gene expression changes.
Methods: The drugs were produced by Boiron Laboratoires, Lyon (F), starting from a whole-plant-hydroalcoholic extract of Gelsemium s. Solutions 1C, 2C, 3C, 4C, 8C and 29C (C= centesimal dilution/dynamization prepared in 30% ethanol/distilled water) were provided in 30-ml glass bottles, wrapped in aluminium foil and were stored in the dark at room temperature in a metal cupboard. Control solutions (“placeboâ€ÂÂ) were serially diluted/dynamized 30% ethanol/distilled water. Before each experiment, 0.05 ml samples of Gelsemium s. and placebo were added to 5 ml of distilled sterile and apyrogenic water in a 15 ml Falcon polystyrene plastic tube, closed and shaken in mechanical shaker DinaA for 7.5 sec (150 strokes) to obtain the final 2C, 3C, 4C, 5C, 9C and 30C succussed dilutions, with ethanol concentration of 0.3% (v/v) (final 0.03% in the assay system).
Human neuroblastoma cell line SHSY5Y was grown in DMEM-F12 medium (Lonza), with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 mg/ml). To assess cell viability and metabolism, 20,000 cells per well were seeded in 96 microplate wells in 200 μl of medium. After overnight incubation, 22 μl of drug or placebo were added and the plate was incubated at 37°C with 5% CO2 in a humidified atmosphere for 72 hours. Then the viability test with reagent WST-1 (Roche) was performed for 3 hours and the absorbance was detected with multiplate reader. A total of 17 experiments, each done with six replicate microwells. To make a relative measurement of protein, the cells were lysed and a Bradford assay was done directly in the plate. The Student t-test and the sign rank test for paired data were utilized for data analysis.
To obtain a profile of gene expression, cells were pre preconditioned with Gelsemium s./placebo dilutions for 24 h, then RNA was isolated and analysed by microarray and RT-PCR. SHSY5Y cells were plated onto Petri dishes (day 1) and the day after the medium was replaced with the medium with 2% FBS (day 2). After 24h, 10%v/v Gelsemium s. or placebo dilutions were added to the medium (day 3) and cells were incubated for a further 24h. On day 4, cells were then harvested and the RNA extracted using the Qiagen RNAeasy Mini Kit following the manufacturer’s instructions. Microarray analysis was performed on a custom 12 x 135 k human NimbleGen microarray containing 45033 genes with 3 probes per target gene. Four biological replicates were analysed for each condition. Analysis of differentially expressed genes was performed using linear modelling and empirical Bayes methods and p-values were adjusted for multiple testing with the Benjamini and Hochberg method. A Human Neurotransmitter Receptors and Regulators RT2 Profiler PCR array (Qiagen) was performed in profiling the expression of genes involved in modulating the biological processes of neurotransmitter biosynthesis, uptake, transport and signaling through neurotransmitter receptors.
Results: In viability tests, cells treated with Gelsemium s. showed slightly higher metabolic activity (3-4 %) than those treated with placebo. Overall comparison of the data for the whole sample of placebo versus that of Gelsemium s. using the Student t-test showed a small but significant difference (p < 0.001). Furthermore, a non parametric approach comparing the two treatments at the same dilution yielded a significant difference under the sign test (p < 0.01) and Wilcoxon rank test for paired data (p < 0.05), so that the values of differences were also considered. The differences between groups having the same dilution (placebo 2C versus Gelsemium s. 2C etc.) were significant in four dilutions: 2 C, 3 C, 4C (p < 0.01), 9 C (p < 0.02), while 5 C and 30 C yielded non-significant values. No changes due to Gelsemium s. were detected using protein assay, suggesting that the viability test revealed effects on metabolic activity instead of on cell proliferation.
In microarray analysis, transcripts expression was analyzed and genes differentially expressed by the Gelsemium s. dilutions were selected. A gene was considered to be differentially expressed if it showed an absolute value of log-ratio greater than or equal to 0.5, an index that translates to a fold-change of 1.4 in transcript quantity. Out of a total of 45,033 transcripts, exposure to Gelsemium s. 2C promoted the selective downexpression of 49 genes (p values adj
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Krivstov, Andrei V., David Twomey, Zhaohui Feng, Matthew C. Stubbs, Todd R. Golub, and Scott A. Armstrong. "MLL-Fusion Proteins Induce a Stem Cell Program in Committed Progenitors To Generate Leukemia Stem Cells." Blood 106, no. 11 (November 16, 2005): 465. http://dx.doi.org/10.1182/blood.v106.11.465.465.
Full textAbstract:
Abstract Leukemias are composed of a hierarchy of cells only a fraction of which have stem cell like properties, and are capable of self-renewal. MLL fusion proteins produced by translocations involving the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 confer stem cell-like properties on committed hematopoietic progenitors. This provides an opportunity to determine if global cellular reprogramming is necessary for leukemia stem cell (LSC) generation from committed progenitors or if induction of a more limited self-renewal signature in committed progenitors is sufficient. We transduced murine IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIhi granulocyte macrophage progenitors (GMPs) with retroviruses encoding the MLL-AF9 fusion protein, which led to the development of acute myelogenous leukemia. From the leukemias we isolated a population of IL-7R− Lin− Sca-1− c-Kit+ CD34int. FcγRII/IIIint. LSCs which can transplant the disease when fewer than 20 cells are injected into secondary recipients. We used hierarchical clustering, K-means clustering and principal component analysis to compare gene expression profiles of the LSC population to the normal lin− sca-1+ c-kit+ HSC-enriched population, IL-7R− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs), IL-7R− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) and GMPs and found that the global gene expression profile most resembles the normal GMP from which they arose. However, a leukemia self-renewal signature was identified that shows significant overlap with a group of genes normally highly expressed in HSCs whose expression decreases during the transition to normal committed progenitors. Supervised analysis and gene set enrichment analysis (GSEA) demonstrated approximately 300 genes in the leukemia self-renewal signature. This is only a subset of the approximately 1500 genes that are highly expressed in the normal HSC-enriched population that show decreased expression in CMPs, MEPs, and GMPs. Next, we determined if this 300-gene leukemia stem cell signature is directly regulated by MLL-AF9 or if there is a hierarchy of gene expression. Assessment of gene expression changes 48 hours after MLL-AF9 expression in isolated GMPs demonstrated increased expression of 23/300 genes in the leukemia self-renewal signature. Of interest, there is a high degree of similarity between the 23 MLL immediate response genes and human MLL-rearranged AMLs including HOXA5, HOXA7, HOXA9, HOXA10, MEIS1 and genes not previously known to have a role in MLL-mediated leukemogenesis such as myocyte enhancer factor 2C (MEF2C). Detailed loss-of-function studies using shRNA and dominant negative mutants show inhibition of MEF2C reduces LSC colony formation and serial replating in semi-solid culture to less than 20% of control. Furthermore shRNA mediated inhibition of MEF2C has a significant impact on proliferation of human MLL-AF9 dependent leukemia cell lines, but not cell lines from other subtypes of AML. These data demonstrate LSCs can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal signature is activated in the process. We have used this program to identify MEF2C as playing a role in MLL-AF9 induced AML. Identification of this program provides an opportunity to further assess its importance in normal tissue homeostasis and neoplastic self-renewal/proliferation, and defines the progression from normal hematopoietic progenitor to leukemia stem cell.
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Bernagozzi, Marco, Nicolas Miché, Anastasios Georgoulas, Cedric Rouaud, and Marco Marengo. "Performance of an Environmentally Friendly Alternative Fluid in a Loop Heat Pipe-Based Battery Thermal Management System." Energies 14, no. 22 (November 18, 2021): 7738. http://dx.doi.org/10.3390/en14227738.
Full textAbstract:
The present investigation aims to devise a thermal management system (TMS) for electric vehicles able to improve on limitations like charging time and all-electric range, together with the safety and environmental impact of the chosen thermal medium. A research gap is identified, as focus is often on addressing system thermal performance without considering that the thermal medium must not only provide suitable performances, but also must not add risks to both passengers and the environment. Thus, this work proposes an innovative cooling system including graphite sheets and a Loop Heat Pipe, filled with Novec™ 649 as working fluid, due to its exceptional environmental properties (GWP = 1 − ODP = 0) and safety features (non-flammable, non-toxic, dielectric). A three-cell module experimental demonstrator was built to compare temperatures when the proposed TMS is run with Novec™ 649 and ethanol. Results of testing over a bespoke fast charge driving cycle show that Novec™ 649 gave a faster start-up and a slightly higher maximum temperature (0.7 °C), meaning that the gains in safety and lower environmental impact brought by Novec™ 649 came without lowering the thermal performance. Finally, the TMS was tested under three different fast charge conditions (1C, 2C, 3C), obtaining maximum temperatures of 28.4 °C, 36.3 °C and 46.4 °C, respectively.
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Kwiatkowska, Monika, Justyna Żabicka, Grzegorz Migdałek, Piotr Żabicki, Marlena Cubała, Jerzy Bohdanowicz, Aneta Słomka, et al. "Comprehensive characteristics and genetic diversity of the endemic Australian Viola banksii (section Erpetion, Violaceae)." Australian Journal of Botany 67, no. 2 (2019): 81. http://dx.doi.org/10.1071/bt18233.
Full textAbstract:
Viola banksii, the type species of section Erpetion, is endemic in eastern mainland Australia. In this paper we characterise morphological and anatomical features and assess genome size and genetic diversity in combination with the breeding system. V. banksii develops exclusively chasmogamous flowers. Ovules are anatropous, crassinucellate and bitegmic, the female gametophyte is of the Polygonum type, and the embryo is of Asterad type surrounded by nuclear endosperm. Pollen is non-heteromorphic, 3-aperturate, and highly viable. V. banksii grows in shade on moist, well drained, often sandy soils, and this is reflected in the anatomy of its organs, which includes a lack of subepidermal collenchyma in aerial parts, large leaf epidermal cells with thin cell walls, a narrow cuticle layer, and vascular bundles with xylem that are not rich in vessels. V. banksii is tolerant to zinc and lead based on phytotoxicity test. The high chromosome number (2n = 10x = 50) does not correspond to a small genome size (2C DNA = 1.27 pg). Low mean intra-populational gene diversity (HS = 0.077) detected by ISSR markers confirms the strong influence of selfing and clonal propagation by pseudostolons. Unique morphological traits of V. banksii include nyctinastic petal movement, the lack of a floral spur, the presence of gland-like protuberances on two stamens, and the presence of pseudostolons, which could be a synapomorphy for the whole section.
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Szuber, Natasha, Terra L. Lasho, Christy Finke, Curtis A. Hanson, Rhett P. Ketterling, Animesh Pardanani, Naseema Gangat, and Ayalew Tefferi. "Determinants of Long-Term Outcome in Type 1/like Calreticulin-Mutated Myelofibrosis." Blood 132, Supplement 1 (November 29, 2018): 1767. http://dx.doi.org/10.1182/blood-2018-99-117458.
Full textAbstract:
Abstract Background: Type 1 calreticulin (CALR) variants comprise ~70% of all CALR mutations in primary myelofibrosis (PMF) and form a distinct phenotypic and prognostic disease subset (Leukemia. 2014;28:1568). Determinants of long-term outcome have not, however, been systematically appraised in this population. The current study documents the natural history, molecular correlates, and independent predictors of overall (OS), leukemia-free (LFS), and thrombosis-free (TFS) survival in CALR type 1/like-mutated myelofibrosis. Methods: Patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were consistent with World Health Organization (PMF, fibrotic/leukemic transformations) (Blood. 2016;127:2391) and International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria (post-essential thrombocythemia (ET) MF) (Leukemia. 2008;22:437). Laboratory and clinical data were retrospectively abstracted corresponding to time of referral (PMF) or myelofibrotic transformation (post-ET MF). Conventional prognostic scoring was as previously outlined (Blood. 2010;115:1703; J Clin Oncol. 2018;36:1769). Recipients of allogeneic stem cell transplant were censored at the time of transplant. Standard statistical methods were used for all analyses using the JMP® Pro 13.0.0 software package (SAS Institute, Cary, NC, USA). Results: A total of 162 consecutive patients with CALR type 1/like-mutated myelofibrosis were identified: 139 (86%) with PMF and 23 (14%) with post-ET MF with median age 55 years (range 23-85), 62% male. The PMF and post-ET MF cohorts displayed similar phenotypic features with the exception of higher platelet counts (median 443 vs 340 x 109/l; P=0.02) in post-ET MF (Table 1). The most frequent co-existing mutations were ASXL1 (n=47; 37%) and SRSF2 (n=4; 3%), with ASXL1 seen more frequently in PMF (39% vs 11% post-ET MF; P=0.07). Over a median follow-up of 6 years (range 0-25 years), a total of 20 (12%) leukemic transformations and 66 (41%) deaths were recorded, with no significant differences between the PMF and post-ET MF cohorts (P=0.16 and 0.11, respectively). Kaplan-Meier survival estimates revealed comparably favorable median OS in both CALR type 1/like-mutated variants: not yet reached and 13 years in post-ET MF vs PMF, respectively (P=0.7) (Figure 1A). Multivariable analysis disclosed moderate to severe sex-adjusted anemia (P<0.001), ≥2% circulating blasts (P<0.001), very high risk (VHR) karyotype (P<0.001), age >70 years (P=0.006), and constitutional symptoms (P=0.008) to be independent predictors of inferior OS in CALR type 1/like-mutated PMF (Table 2). LFS was significantly shortened in the presence of IDH1 mutations (P=0.01) and platelets <100 x 109/l (P=0.02) while IDH2 mutations (P=0.02), leukocytosis ≥11 x 109/l (P=0.03) and history of arterial thrombosis (P=0.04) were independent predictors of shortened TFS. Importantly, myelofibrosis variant (primary vs post-ET) did not influence survival (P=0.7) or complication rates (P=0.8 for LFS, P=0.09 for TFS) (Table 2). The karyotype- and mutation-enhanced international prognostic scoring system (MIPSS70+ version 2.0), was effective in risk stratifying type 1/like CALR-mutated PMF (P-values 0.01 to <0.0001), with the exception of low vs intermediate (P=0.22) and intermediate vs high risk (P=0.49) (Figure 1C). The detrimental influences of unfavorable/VHR karyotype and ASXL1 mutations were confirmed (Figure 2A-B) while borderline adverse and prognostically neutral effects were seen on OS for U2AF1 (n=116; P=0.05) and SRSF2 (n=120; P=0.98) respectively (Figure 2C-D). Conclusions: The current study documents analogous disease patterns in primary and post-ET CALR type 1/like-mutated MF and provides information on determinants of long-term survival. Disclosures No relevant conflicts of interest to declare.
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Kawamoto, Manabu, Akiko Fujiwara, Shin-ichi Kuno, and Ikuo Yasumasu. "Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development." Zygote 8, S1 (December 1999): S68—S69. http://dx.doi.org/10.1017/s0967199400130370.
Full textAbstract:
Serine/threonine protein phosphatases expected to participate in the process of signal transduction, cell movement such as cell division and gene expression (Kinoshita et al., 1990; Healy et al., 1991; Mayer-Jaekel et al., 1993; Mumby & Walter, 1993), are classified into type 1 (PP1), type 2A (PP2A), type 2B and type 2C in mammalian cells. PP1 and PP2A are known to be strongly inhibited by okadaic acid (OA) (Tachibana et al., 1981; Bialojan Takai, 1988), a polyether fatty acid isolated from the marine sponge Halicondria okadai (Haystead et al., 1989). OA is also known to inhibit PP2A at lower concentrations than that to block PP1 in mammalian cells, but does not inhibit the activities of other phosphatase species (Ishihara et al., 1989).The p-nitrophenyl phosphate (pNPP) splitting activity in the extract obtained from eggs of the sea urchin Hemicentrotus pulcherrimus was found to be inhibited by OA and calyculin A (CLA), potent inhibitors of PP1 and PP2A. OA-sensitive phosphatases are known to catalyse pNPP splitting (Takai & Mieskes, 1991), in the same manner as other OA-insensitive phosphatases.Four peaks of the pNPP splitting activity were obtained by QAE-Toyopearl chromatography in the extract of sea urchin eggs. In two of these four peaks, pNPP splitting reactions were strongly inhibited by OA and CLA at quite low concentration. High sensitivities of the pNPP splitting reaction to OA and CLA in these two peaks suggest that pNPP splitting results from the reaction catalysed by PP2A. The molecular masses of proteins exhibiting OA-sensitive pNPP splitting activities in these two peaks were found to be about 160 kDa by Superdex 200HR, and were similar to that of mammalian PP2A trimeric holoenzyme. By immunoblot analyses with anti-human PP2A catalytic subunit antibody, an immunoreactive 36 kDa protein was found by SDS-PAGE in a peak of OA-sensitive pNPP splitting activity obtained by QAE-Toyopearl chromatography. Sea urchin eggs have at least two PP2A-like enzymes with similar molecular masses to that of mammalian PP2A, and one of them contains human-type catalytic subunit.
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Kai, Wenbin, Juan Wang, Bin Liang, Ying Fu, Yu Zheng, Wenbo Zhang, Qian Li, and Ping Leng. "PYL9 is involved in the regulation of ABA signaling during tomato fruit ripening." Journal of Experimental Botany 70, no. 21 (August 31, 2019): 6305–19. http://dx.doi.org/10.1093/jxb/erz396.
Full textAbstract:
Abstract Abscisic acid (ABA) regulates fruit ripening, yet little is known about the exact roles of ABA receptors in fruit. In this study, we reveal the role of SlPYL9, a tomato pyrabactin resistance (PYR)/pyrobactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) protein, as a positive regulator of ABA signaling and fruit ripening. SlPYL9 inhibits protein phosphatase-type 2C (PP2C2/6) in an ABA dose-dependent way, and it interacts physically with SlPP2C2/3/4/5 in an ABA-dependent manner. Expression of SlPYL9 was observed in the seeds, flowers, and fruits. Overexpression and suppression of SlPYL9 induced a variety of phenotypes via altered expression of ABA signaling genes (SlPP2C1/2/9, SlSnRK2.8, SlABF2), thereby affecting expression of ripening-related genes involved in ethylene release and cell wall modification. SlPYL9-OE/RNAi plants showed a typical ABA hyper-/hypo-sensitive phenotype in terms of seed germination, primary root growth, and response to drought. Fruit ripening was significantly accelerated in SlPYL9-OE by 5–7 d as a result of increased endogenous ABA accumulation and advanced release of ethylene compared with the wild-type. In the SlPYL9-RNAi lines, fruit ripening was delayed, mesocarp thickness was enhanced, and petal abscission was delayed compared with the wild-type, resulting in conical/oblong and gourd-shaped fruits. These results suggest that SlPYL9 is involved in ABA signaling, thereby playing a role in the regulation of flower abscission and fruit ripening in tomato.
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Deng, Yilun, Harshita B. Gupta, Justin Michael Drerup, Ryan Michael Reyes, Jenny Mendez, Xinyue Zhang, Alvaro S. Padron, Myrna Garcia, and Tyler J. Curiel. "CD122-selective IL-2 complexes treat ovarian carcinomas and melanoma, alter Treg differentiation and induce more CD8+CXCR5+TCF-1+ stem T cells when combined with αPD-L1." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 136.15. http://dx.doi.org/10.4049/jimmunol.202.supp.136.15.
Full textAbstract:
Abstract Anti-tumor T cells (Teffs) engage IL2 largely through CD122, vs. CD25 for regulatory T cells (Tregs). In ID8agg ovarian cancer (OC), CD122-selective IL2 complexes (IL2c, made from CD122-blocking ab+IL-2), increased Teff numbers and functions as expected but unexpectedly reduced tumor Treg function, with near-complete tumor rejection in OC. IL2c reduced lineage specific molecules (e.g., Tbet) and cytokines (e.g., IFNg) in tumor Tregs, suggesting IL2c improves Treg differentiation, but not explaining IL2c-driven Treg defects. In vitro, IL-2 and IL-2c produced similar Treg numbers and Foxp3 from naïve CD4+T cells, with functional data pending. αPD-L1 improved IL2c efficacy for full tumor rejection in OC and B16 melanoma. Increased CD8+CXCR5+PD1+Tim3−TCF-1+stem-like T cells (CXCR5+) are associated with favorable αPD-1 or αPD-L1 outcome in mice and humans. IL2c + αPD-L1 markedly increased CXCR5+in B16 melanoma and to a lesser extent in OC. IL2c + αPD-L1 increased tumor CXCR5+perforin expression in B16 vs. either single agent. Injecting tumor-infiltrating CXCR5+into RagKO mice produced CXCR5+Tim3− and CXCR5−Tim3+cells confirming tumor CXCR5+stem cell properties. These data demonstrate several novel IL2c mechanisms to exploit in distinct cancers and treatment combinations. Ongoing work tests IL2c, αPD-L1 and combo treatment effects on CXCR5+ stemness and mechanisms for CXCR5+ induction, and mechanisms for IL2c-driven Treg dysfunction.
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Gao, Caiyun, Miao Deng, Xiaoming Yang, Wanwen Yu, Jinfeng Cai, Yuanbao Shi, Zhibo Zhu, et al. "Genome-Wide Identification and Coexpression Network Analysis of DNA Methylation Pathway Genes and Their Differentiated Functions in Ginkgo biloba L." Forests 11, no. 10 (October 9, 2020): 1076. http://dx.doi.org/10.3390/f11101076.
Full textAbstract:
DNA methylation plays a vital role in diverse biological processes. DNA methyltransferases (DNMTs) genes and RNA-directed DNA methylation (RdDM)-related genes are key genes responsible for establishing and maintaining genome DNA methylation in plants. In the present study, we systematically identified nine GbDNMTs in Ginkgo biloba, including the three common families of GbMET1a/1b, GbCMT2, and GbDRMa/b/2a/2b/2c, and a fourth family—GbDNMT3—which is absent in most angiosperms. We also identified twenty RdDM-related genes, including four GbDCLs, six GbAGOs, and ten GbRDRs. Expression analysis of the genes showed the different patterns of individual genes, and 15 of 29 genes displayed expression change under five types of abiotic stress. Gene coexpression analysis and weighted gene co-expression network analysis (WGCNA) using 126 public transcriptomic datasets revealed that these genes were clustered into two groups. In group I, genes covered members from all six families which were preferentially expressed in the ovulate strobile and fruit. A gene ontology (GO) enrichment analysis of WGCNA modules indicated that group I genes were most correlated with the biological process of cell proliferation. Group II only consisted of RdDM-related genes, including GbDRMs, GbAGOs, and GbRDRs, but no GbDCLs, and these genes were specifically expressed in the cambium, suggesting that they may function in a dicer-like (DCL)-independent RdDM pathway in specific tissues. The gene module related to group II was most enriched in signal transduction, cell communication, and the response to the stimulus. These results demonstrate that gene family members could be conserved or diverged across species, and multi-member families in the same pathway may cluster into different modules to function differentially. The study provides insight into the DNA methylation genes and their possible functions in G. biloba, laying a foundation for the further study of DNA methylation in gymnosperms.
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Watanabe, Yukina, Yasunobu Kato, and Toshiyuki Yamamoto. "Diltiazem-associated, photo-distributed hyperpigmentation in a patient with Sjögren’s syndrome." Our Dermatology Online 13, no. 4 (October 1, 2022): 471–72. http://dx.doi.org/10.7241/ourd.20224.33.
Full textAbstract:
Sir, Herein, we report a case of hyperpigmented macules on ultraviolet exposed areas during the intake of diltiazem hydrochloride (DTH) in a patient with Sjögren’s syndrome. A 63-year-old Japanese female with a history of Sjögren’s syndrome and vasospastic angina presented to our hospital with brown, irregular macules on the neck, face, and both hands, which she first noticed one year earlier. The patient had a history of taking DTH for vasospastic angina for the previous fifteen years. A physical examination revealed pale brown macules and plaques irregularly scattered around the corners of the mouth, lower lip, and neck (Figs. 1a and 1b). In addition, there were numerous erythematous papules on the dorsal side of both hands. The oral mucosal membrane, scalp, and nails were not involved. A biopsy specimen taken from the dorsal side of a hand revealed individual cell keratinization in the epidermis, intercellular edema, liquefaction degeneration of the basal layer, and band-like lymphocyte infiltrate with melanophages in the superficial dermis (Fig. 2a). Immunohistochemistry revealed the infiltration of CD3+, CD4+, CD8+, and HLA-DR+ lymphocytes in the superficial dermis (Figs. 2b and 2c). Routine laboratory examinations of the blood cell count, serum, and urine showed no abnormalities. Other tests showed positive antinuclear antibody (1:80, nucleolar) and anti-SS-A antibody (240 U/mL; normal: < 9.9), whereas hepatitis C virus antibody, rheumatoid factor, anti-CCP antibody, and anti-SS-B antibody were negative. DTH was switched to another medicine, and ascorbic acid, calcium pantothenate, and topical steroids were initiated. In addition, we advised the patient to avoid sun exposure. Although no improvement was observed in the rash in the first four months, it disappeared at follow-up several years later (Figs. 3a and 3b).
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Chen, Yunfang, Sheng Wang, Xin Fu, Wenqu Zhou, Wei Hong, Dongting Zou, Xichong Li, Jinbao Liu, Pixin Ran, and Bing Li. "tert-Butylhydroquinone mobilizes intracellular-bound zinc to stabilize Nrf2 through inhibiting phosphatase activity." American Journal of Physiology-Cell Physiology 309, no. 3 (August 1, 2015): C148—C158. http://dx.doi.org/10.1152/ajpcell.00031.2015.
Full textAbstract:
The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular “free” zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation.
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Koralewski, T. E., J. E. Brooks, and Konstantin V. Krutovsky. "Molecular evolution of drought tolerance and wood strength related candidate genes in loblolly pine (Pinus taeda L.)." Silvae Genetica 63, no. 1-6 (December 1, 2014): 59–66. http://dx.doi.org/10.1515/sg-2014-0009.
Full textAbstract:
Abstract Loblolly pine (Pinus taeda L.) is an intensely studied species that has become a model system for conifers. It is one of the most important commercial crops in the southeastern United States and grows across a vast territory. Due to exposure to this current diverse environment and the fluctuating climatic conditions of the past, it has likely accumulated substantial variation in adaptive trait and wood strength related genes. We merged a set of newly collected and previously published genomic DNA sequence data and analyzed them for departures from neutrality in 32 drought tolerance and wood strength related candidate genes using neutrality tests, such as Tajima’s D, HKA, MK and nonsynonymous to synonymous substitutions ratio (Z-test). Three other major Southern pines closely related to P. taeda (Pinus echinata Mill., P. elliottii Engelm., and P. palustris Mill.) were used as outgroups in interspecific tests. In three loci (4-coumarate: CoA ligase, putative cell-wall protein and trans-cinnamate 4-hydroxylase 2) neutrality was rejected by both intra- and interspecific tests, consistent with purifying selection. Neutrality was also rejected in several other loci (alpha-tubulin, arabinogalactan 4, arabinogalactan 6, cinnamate 4-hydroxylase 1, cinnamoyl CoA reductase, cinnamyl alcohol dehydrogenase, caffeoyl CoA O-methyltransferase 1, early response to drought 3, glycine hydroxymethyltransferase, ABI1 protein phosphatase 2C-like, putative wall-associated protein kinase, and unknown gene ug_2-498); however, these results are difficult to interpret because only one of the tests proved significant. This study contributes to the ongoing discussion about natural selection in putative adaptive genes in loblolly pine. However, unambiguous interpretation of the results often remains problematic.