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Klein, William H., and Xiaotao Li. "Function and Evolution of Otx Proteins." Biochemical and Biophysical Research Communications 258, no. 2 (May 1999): 229–33. http://dx.doi.org/10.1006/bbrc.1999.0449.
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Klein, William H., and Xiaotao Li. "Function and Evolution of Otx Proteins." Biochemical and Biophysical Research Communications 260, no. 2 (July 1999): 575. http://dx.doi.org/10.1006/bbrc.1999.0938.
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Acampora, D., V. Avantaggiato, F. Tuorto, and A. Simeone. "Genetic control of brain morphogenesis through Otx gene dosage requirement." Development 124, no. 18 (September 15, 1997): 3639–50. http://dx.doi.org/10.1242/dev.124.18.3639.
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Understanding the genetic mechanisms that control patterning of the vertebrate brain represents a major challenge for developmental neurobiology. Previous data suggest that Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, might both contribute to brain morphogenesis. To gain insight into this possibility, the level of OTX proteins was modified by altering in vivo the Otx gene dosage. Here we report that Otx genes may cooperate in brain morphogenesis and that a minimal level of OTX proteins, corresponding either to one copy each of Otx1 and Otx2, or to only two copies of Otx2, is required for proper regionalization and subsequent patterning of the developing brain. Thus, as revealed by anatomical and molecular analyses, only Otx1−/−; Otx2+/− embryos lacked mesencephalon, pretectal area, dorsal thalamus and showed an heavy reduction of the Ammon's horn, while the metencephalon was dramatically enlarged occupying the mesencencephalic area. In 8.5 days post coitum (d.p.c.) Otx1−/−; Otx2+/− embryos, the expression patterns of mesencephalic-metencephalic (mes-met) markers such as En-1 and Wnt-1 confirmed the early presence of the area fated to give rise to mesencephalon and metencephalon while Fgf-8 transcripts were improperly localized in a broader domain. Thus, in Otx1−/−; Otx2+/− embryos, Fgf-8 misexpression is likely to be the consequence of a reduced level of specification between mes-met primitive neuroepithelia that triggers the following repatterning involving the transformation of mesencephalon into metencephalon, the establishment of an isthmic-like structure in the caudal diencephalon and, by 12.5 d.p.c., the telencephalic expression of Wnt-1 and En-2. Taken together these findings support the existence of a molecular mechanism depending on a precise threshold of OTX proteins that is required to specify early regional diversity between adjacent mes-met territories and, in turn, to allow the correct positioning of the isthmic organizer.
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Kemper, Ryan M., Manfred Meng, and Daniel J. Crona. "Abstract B030: Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer." Cancer Research 82, no. 23_Supplement_2 (December 1, 2022): B030. http://dx.doi.org/10.1158/1538-7445.cancepi22-b030.
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Abstract Background: Bladder cancer (BC) remains a common and deadly malignancy, with a projected 81,180 new diagnoses and 17,100 deaths in the United States in 2022. According to the MSK/TCGA Bladder Cancer dataset, mutations in DNA damage response (DDR) genes that are part of the homologous recombination (HR) pathway occurred in up to 55% of patients, representing a potential therapeutic target in BC. In previous work, we used UNC’s EpiG Diamond compound library to show that, as a class, inhibition of the methyl-lysine reader bromodomain and extra-terminal domain (BET) proteins potently abrogate BC cell line viability. Here, we evaluated mechanisms related to HR inhibition that could explain pan-BET inhibitor OTX-015’s potency in BC. Methods: 5637 and J82 BC cells were treated with 1 µM OTX-015 or 0.1% DMSO control for 48-hours, total RNA was extracted, and then bulk RNA-sequencing (RNA-seq) was performed on an Illumina NovaSeq 6000 (Novogene, Sacramento, CA). Expression data was analyzed using Geneious Prime v2022.2.1 (Biomatters, San Diego, CA). Cells were then treated with 1 μM OTX-015, 5 μM of the PARP inhibitor olaparib, or combination for gene and protein expression analysis. Gene expression changes in BRCA1, BRCA2, PALB2, and RAD51, as well as MYC as a positive control, were confirmed by RT-PCR in biological triplicates after 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. Western blotting evaluated OTX-015 and olaparib effects on BRCA1, BRCA2, c-MYC, PALB2, and RAD51 protein expression after 72-hour incubation, using a GAPDH loading control and compared to a 0.1% DMSO control. Last, cells were treated with the same treatment schema for 24-72 hours, fixed with methanol, stained with propidium iodide (25 μg/mL), and flow cytometry evaluated effects on cell cycle using a ThermoFisher Attune NxT and FlowJo v10.8.1 (BD Life Sciences, Ashland, OR). Results: Bulk RNA-seq analyses revealed significantly altered expression of HR pathway genes in 5637 and J82 cells treated with OTX-015±olaparib. In 5637 cells, OTX-015 alone significantly reduced BRCA1, BRCA2, PALB2 and RAD51 expression, (n=3; P<0.001 for all), but only PALB2 and RAD51 expression was significantly reduced after combination treatment (n=3; P<0.001 for both). In J82 cells, OTX-015 alone and combination treatment significantly reduced BRCA1, PALB2 and RAD51 expression, (n=3; P<0.001 for all). Similarly, both OTX-015 and combination treatment resulted in reduced BRCA1, c-MYC, PALB2 and RAD51 protein expression in both cell lines. In both cell lines, combination treatment resulted in a substantially increased G2/M fraction at 48 hours compared to 0.1% DMSO (5637 cells: 62.9% vs 12.6% G2/M; J82 cells: 28.2% vs 17.8% G2/M). Conclusions: These data indicate that OTX-015-mediated inhibition of RAD51 and PALB2 could be responsible for impaired HR. Cell cycle data revealed a G2/M stall, rather than an S phase stall, suggesting mechanisms independent from impaired HR could be responsible for cell cycle dysregulation. Citation Format: Ryan M. Kemper, Manfred Meng, Daniel J. Crona. Combined inhibition of BET proteins and PARP promotes impaired DNA damage repair response and cell cycle dysregulation in preclinical models of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B030.
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Kemper, Ryan M., Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, and Daniel James Crona. "Abstract 3267: ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3267. http://dx.doi.org/10.1158/1538-7445.am2022-3267.
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Abstract Background: Bladder cancer (BC) is a common and deadly disease. Inactivating ARID1A mutations occur in up to 30% of metastatic BC tumors, making it the most commonly mutated epigenetic gene and the 4th most commonly mutated gene overall in BC. ARID1A mutations are associated with decreased response to therapy and poor prognosis; thus, there is a need to develop therapies specifically targeting ARID1A mutant tumors. Using the UNC EpiG Diamond compound library, a set of well-annotated small molecule probes targeting chromatin regulatory proteins, we determined that inhibitors of bromodomain and extraterminal (BET) proteins potently inhibit the viability of BC cells. Here, we tested the hypothesis that ARID1Amut cells are particularly sensitized to the pan-BET inhibitor OTX-015. Methods: AR1D1A-competent (ARID1AWT) 5637 and ARID1A-mutated (ARID1Amut) HT1197 cells were treated with eight ascending concentrations of OTX-015 (0.1 nM-100 µM), and cell viability was measured using CellTiter-Glo™ after 72-120-hour incubations. IC50 values were calculated using a four-parameter non-linear regression model. Gene expression of BET inhibitor target genes, MYC, ARID1B, and RAD51, were evaluated by RT-PCR after a 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. CellTiter-Glo™ and RT-PCR experiments were conducted in technical and biologic triplicates. Western blotting evaluated OTX-015 effects on c-MYC, BAF250B (encoded by ARID1B), and RAD51 protein expression after 72-hour incubation. Results: OTX-015 was 8-times more potent in ARID1Amut HT1197 cells at 120 h than in ARID1AWT 5637 cells (IC50: 0.12 μM vs. 1.0 μM). OTX-015 treatment (1 µM) significantly reduced ARID1B and RAD51 mRNA expression versus 0.1% DMSO control (83% and 86% reduction, respectively, both P<0.0001). At the same concentration, OTX-015 did not significantly reduce MYC mRNA expression HT1197 cells (25% reduction, P=0.31), but did significantly reduce MYC expression in 5637 cells (57% reduction, P<0.0001) at 48 h. When comparing HT1197 to 5637 cells, OTX-015 treatment (1 µM) caused a greater reduction in ARID1B mRNA expression (83% vs. 62% reduction, P=0.02) and RAD51 mRNA expression (86% vs. 57%, P=0.001) at 48 h. Finally, OTX-015 treatment (1 µM) also resulted in more dramatically reduced c-MYC, ARID1B and RAD51 protein expression in HT1197 cells at 72 h, when compared to 5637 cells. Conclusions: These preliminary results support future lines of inquiry into the molecular mechanisms that underlie sensitization of ARID1Amut cells to BET protein inhibition. Citation Format: Ryan M. Kemper, Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, Daniel James Crona. ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3267.
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Acampora, Dario, Massimo Gulisano, and Antonio Simeone. "Genetic and molecular roles of Otx homeodomain proteins in head development." Gene 246, no. 1-2 (April 2000): 23–35. http://dx.doi.org/10.1016/s0378-1119(00)00070-6.
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Senapedis, William, Elmer Figueroa, Kayleigh Gallagher, Jeremiah Farelli, Robert Lyng, Charles O'Donnell, Joseph Newman, and Thomas McCauley. "Abstract 2629: Epigenetic modulation of the MYC oncogene as a potential novel therapy for HCC." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2629. http://dx.doi.org/10.1158/1538-7445.am2022-2629.
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Abstract Hepatocellular carcinoma (HCC), the fourth leading cause of cancer deaths, represents an unmet medical need with few therapeutic options. Sorafenib has been used as a systemic therapy for HCC for >10 years but patients frequently develop resistance with oncogenic c-MYC (MYC) identified as a correlating prognostic factor. MYC over-expression is associated with aggressive disease in up to ~70% of HCC. While MYC represents an attractive therapeutic target, it has historically been considered undruggable, largely because it lacks a structured binding pocket and its expression is tightly autoregulated. The MYC gene and its regulatory elements are part of an insulated genomic domain (IGD), a chromatin looping region anchored by CTCF. Here we describe our approach to specifically modulate levels of MYC expression by utilizing targeted mRNA-encoded proteins, Omega Epigenomic Controllers (OECs), to mediate epigenetic regulation while potentially overcoming MYC autoregulation. For screening, putative OECs were directed to 2 loci on the MYC IGD. Identified target loci were used to design optimized OECs, including development candidate OTX-2002. We characterized OTX-2002 in HCC cell lines, measuring MYC mRNA and cell viability. OTX-2002 was tested for durable epigenetic and transcriptomic changes. Changes in MYC protein levels and pathway signaling were measured using proteomic methods. Finally, we analyzed activity of OTX-2002 in in vivo subcutaneous (subQ) and orthotopic HCC models by assessing tumor volume, tumor-associated bioluminescence (BLI) and immunohistochemistry (IHC). OTX-2002 was effective at decreasing MYC mRNA, protein and cell viability in HCC cells while sparing normal cells. In HCC cells, OTX-2002 median EC50 of inhibition is <0.001 ng/mL for MYC mRNA and 120 ng/mL for cell viability. Importantly, the effects of OTX-2002 persisted for >2 weeks, providing durable MYC mRNA repression. IV delivery of OTX-2002 in lipid nanoparticles at 3 and 6 mg/kg Q5D in a Hep 3B subQ model in athymic nude mice demonstrated statistically significant tumor growth inhibition (TGI) of 54% and 63%, respectively, by Day 23 compared to negative control. OTX-2002-treated mice did not have a significant decrease in bodyweight (BW) compared to negative control or sorafenib-treated mice. IHC of OTX-2002 and control treated tumors showed significant down-regulation of MYC with reduced proliferation (Ki67) and increased apoptosis (Caspase 3). In a Hep 3B orthotopic model 3 mg/kg OTX-2002 Q5D showed a comparable reduction of BLI to sorafenib at 50 mg/kg QD without the reduction in BW. Our findings identify a therapeutic in vitro and in vivo approach that enables transcriptional modulation of the MYC oncogene through precise epigenomic programming of the IGD in which it resides. Targeting MYC in this manner may represent a potentially differentiated and viable approach to the treatment of HCC in humans. Citation Format: William Senapedis, Elmer Figueroa, Kayleigh Gallagher, Jeremiah Farelli, Robert Lyng, Charles O'Donnell, Joseph Newman, Thomas McCauley. Epigenetic modulation of the MYC oncogene as a potential novel therapy for HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2629.
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Bellipanni, Gianfranco, Tohru Murakami, O. Geoffrey Doerre, Peter Andermann, and Eric S. Weinberg. "Expression of Otx Homeodomain Proteins Induces Cell Aggregation in Developing Zebrafish Embryos." Developmental Biology 223, no. 2 (July 2000): 339–53. http://dx.doi.org/10.1006/dbio.2000.9771.
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Martin, Richard, Virginie Sanguin-Gendreau, Mathieu Tremblay, Elena Levantini, Christina Magli, and Trang Hoang. "Regulation of SCL Expression by the Homeodomain Protein Otx-1 and the Erythroid Transcription Factor GATA-1." Blood 104, no. 11 (November 16, 2004): 1598. http://dx.doi.org/10.1182/blood.v104.11.1598.1598.
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Abstract Members of the bicoid homeodomain-containing proteins are important in establishing left-right asymmetry and the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that Otx1, a member of the bicoid homeodomain-containing proteins, is co-expressed with the SCL transcription factor in hematopoietic pluripotent and erythroid progenitor cells and Otx1-deficiency impairs the erythroid compartment in mice, associated with decreased SCL levels. In the present study, we provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. First, we show by chromatin immunoprecipitation that Otx1 and GATA-1 are specifically bound to the SCL proximal promoter in erythroid cells. Second, Otx-1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity depends on the integrity of the proximal GATA site of the SCL promoter 1a. At the molecular level, we show that this synergy occurs via a physical interaction between Otx-1 and GATA-1 in erythroid cells, which maps to the homeodomain of Otx-1. Furthermore, a gain of function of Otx1 in primary hematopoietic cells gives rise to a 6-fold increase in endogenous SCL levels, an increase in TER119-positive erythroid cells and a decrease in the number of CD11b-positive myeloid cells. Finally, a gain of function of SCL rescues the erythroid deficiency in Otx1−/− mice, consistent with the view that SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL operate within the same genetic pathway to specify the erythroid fate during hematopoiesis.
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Li, Xiaotao, Chin-Kai Chuang, Chai-An Mao, Lynne M. Angerer, and William H. Klein. "Two Otx Proteins Generated from Multiple Transcripts of a Single Gene inStrongylocentrotus purpuratus." Developmental Biology 187, no. 2 (July 1997): 253–66. http://dx.doi.org/10.1006/dbio.1997.8610.
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Hinman, Veronica F., Albert T. Nguyen, and Eric H. Davidson. "Expression and function of a starfish Otx ortholog, AmOtx: a conserved role for Otx proteins in endoderm development that predates divergence of the eleutherozoa." Mechanisms of Development 120, no. 10 (October 2003): 1165–76. http://dx.doi.org/10.1016/j.mod.2003.08.002.
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Rizzitano, Sara, Alessandra Cavanè, Marco Piazzoni, Antonio Vendramin, Silvia Gimondi, Giulia Biancon, Sofia Cannara Malan, Anna Dodero, Paolo Corradini, and Cristiana Carniti. "Synergistic Anti-Tumor Efficacy of BET Inhibitors JQ1 and Otx-015 in Combination with Dasatinib in Preclinical Models of T-Cell Lymphomas." Blood 128, no. 22 (December 2, 2016): 3967. http://dx.doi.org/10.1182/blood.v128.22.3967.3967.
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Abstract Background: Approximately 50% of patients with peripheral T-cell lymphoma (PTCL) enter long-term remission after standard chemotherapy and stem cell transplantation. Patients who do not respond to chemotherapy have few treatment options highlighting the critical need for new effective and targeted therapeutics. Aberrant T cell receptor (TCR) and tyrosine kinase (TK) signaling have been described in PTCL (Agostinelli 2014;Netchiporouka 2014). Single-agent TK inhibitors (TKIs) have significantly improved patient outcomes across multiple tumor subtypes. However, TKI therapy is rarely curative. The recent discovery of a subgroup of PTCL characterized by high levels of GATA3 and c-Myc expression and poor prognosis (Iqbal 2014; Manso 2016), establishes the rationale of targeting c-Myc in PTCLs. Based on the demonstration that pharmacologic inhibition of c-Myc is achievable through targeting bromodomain and extra terminal (BET) family of chromatin adapters, the therapeutic potential of BET inhibition was assessed in a panel of T cell lymphoma and leukemia cell lines. Since expression of c-Myc is regulated by the TCR, we also hypothesized that simultaneous targeting of c-Myc and TCR would significantly enhance the antiproliferative effects of BET inhibitors (BETis) and TKI alone in preclinical models of PTCL. Methods: Five T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR-2, Karpas 299, Sup-T1, HH) were incubated with escalating doses of JQ1 (a small-molecule BETi with the highest affinity for BRD4) and OTX-015 (a BETi with a broader affinity for BRD2, BRD3, BRD4) and the tyrosine-kinase-inhibitor Dasatinib. Analysis of cell viability, cell cycle distribution, apoptosis and mitochondrial depolarization was performed using flow cytometry. Effects of treatments were assessed using gene expression profiling (GEP) and western blotting (WB). Combinations were evaluated using the Chou-Talalay Combination Index (CI), calculated with CompuSyn software (CompuSyn Inc, Paramus, NJ). Results: JQ1 and OTX-015 show antiproliferative activity with IC50 at nanomolar concentrations in all cell lines. As assessed determining viable cells by PI exclusion and flow cytometry, JQ1 and OTX-015 are similarly active in a dose-dependent manner in all cell lines. To understand the activity of JQ1 and OTX-015, we analyzed cell-cycle distribution using flow cytometry. JQ1 and OTX-015 induce a cell cycle arrest with G1-phase accumulation and decrease S-phase with the exception of SUPT1 cells that are characterized by a cell cycle arrest in G2-phase. Minimal increase in the sub-G1 population is observed in all cell lines, suggesting that JQ1 and OTX-015 mainly exert a cytostatic effect. We then examined GATA3 and c-Myc protein levels in all cell lines: varying amounts of GATA3 and c-Myc proteins were observed but a strong correlation between GATA3 and c-Myc expression was detected. After JQ1 and OTX-015 exposure, c-Myc protein level decrease in all cell lines apart from SUP-T1 cell line. Here c-Myc level do not change significantly upon BETis exposure, suggesting that BETis target other pathways relevant for SUP-T1 survival. Dasatinib efficiently inhibits the proliferation in all cell lines at micromolar concentrations in a dose-dependent manner. Dasatinib induces G0/G1-phase arrest and an increase in sub-G1 population indicating a modest induction of apoptosis confirmed by caspase-9 activation and mitochondrial depolarization. Compared to all single agents, combined treatments with sub-optimal concentrations of Dasatinib and JQ1 or OTX-015 exert synergistic lethal activity against all tested cell lines (C.I.<1). To uncover the main biological processes behind the synergistic interactions of BETis and Dasatinib, cell cycle analysis was assessed indicating that both combinations induce a significant increase of sub-G1 population associated with massive mitochondrial depolarization and cleavage of Caspase-9 and PARP. Conclusions: The experiments presented here support the combination of BET inhibitors with the TK inhibitor Dasatinib for PTCLs. Our data suggest a synergistic interaction for the combination of both BETis and Dasatinib in vitro. Mechanistically, combined treatments exert synergistic anti-tumor effects in all cell lines through growth inhibitory effects, direct induction of cell death by promotion of caspase-dependent apoptosis and mitochondrial depolarization. Disclosures No relevant conflicts of interest to declare.
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Bach, I., C. Carriere, H. P. Ostendorff, B. Andersen, and M. G. Rosenfeld. "A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins." Genes & Development 11, no. 11 (June 1, 1997): 1370–80. http://dx.doi.org/10.1101/gad.11.11.1370.
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Mancini, Pamela, Michele Castelli, and Robert Vignali. "Identification and evolution of molecular domains involved in differentiating the cement gland-promoting activity of Otx proteins in Xenopus laevis." Mechanisms of Development 130, no. 11-12 (November 2013): 628–39. http://dx.doi.org/10.1016/j.mod.2013.09.002.
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Goriely, A., M. Stella, C. Coffinier, D. Kessler, C. Mailhos, S. Dessain, and C. Desplan. "A functional homologue of goosecoid in Drosophila." Development 122, no. 5 (May 1, 1996): 1641–50. http://dx.doi.org/10.1242/dev.122.5.1641.
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We have cloned a Drosophila homologue (D-gsc) of the vertebrate homeobox gene goosecoid (gsc). In the Gsc proteins, the pressure for conservation has been imposed on the homeodomain, the functional domain of the protein: sequence homology is limited to the homeodomain (78% identity) and to a short stretch of 7 aminoacids also found in other homeoproteins such as Engrailed. Despite this weak homology, D-gsc is able to mimic gsc function in a Xenopus assay, as shown by its ability to rescue the axis development of a UV-irradiated embryo. Moreover, our data suggest that the position of insect and vertebrate gsc homologues within a regulatory network has also been conserved: D-gsc expression is controlled by decapentaplegic, orthodenticle, sloppy-paired and tailless whose homologues control gsc expression (for BMP4 and Otx-2), or are expressed at the right time and the right place (for XFKH1/Pintallavis and Tlx) to be interacting with gsc during vertebrate development. However, the pattern of D-gsc expression in ectodermal cells of the nervous system and foregut cannot easily be reconciled with that of vertebrate gsc mesodermal expression, suggesting that its precise developmental function might have diverged. Still, this comparison of domains of expression and functions among Gsc proteins could shed light on a common origin of gut formation and/or on basic cellular processes. The identification of gsc target genes and/or other genes involved in similar developmental processes will allow the definition of the precise phylogenetic relationship among Gsc proteins.
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Lam, Fred. "CLRM-04. PHASE I/II SAFETY AND EFFICACY STUDY OF BET BROMODOMAIN INHIBITOR OTX-015 WITH OLAPARIB AND LOMUSTINE IN PATIENTS WITH RECURRENT GLIOBLASTOMA." Neuro-Oncology Advances 3, Supplement_4 (September 21, 2021): iv1—iv2. http://dx.doi.org/10.1093/noajnl/vdab112.003.
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Abstract Standard of care for patients with glioblastoma (GBM) includes resection with concurrent temozolomide (TMZ) and radiotherapy, with inevitable disease recurrence. Upon recurrence, tumors are often resistant to first-line therapies and/or have infiltrated eloquent or deep brain regions, precluding repeat resection. There is currently no standard of care for recurrent GBM and patients succumb to their disease burden within 12- 15 months of their initial diagnosis of recurrence, exposing an unmet need to find novel therapies to treat recurrent disease. Bromodomain and extraterminal (BET) proteins are chromatin readers that affect transcription of genes. The oral BET inhibitor (BETi) OTX-015 has shown promise in a dose-escalation, phase I study in patients with acute leukemia and other BET inhibitors are currently in phase I studies for the treatment of primary brain tumors. We have recently shown that BET inhibition increases DNA damage and mitotic catastrophe in oncogenic cells by increasing transcription-replication conflicts and downregulating expression of key DNA damage checkpoint proteins, and have also shown its efficacy in decreasing tumor burden and improving survival when combined with TMZ in intracranial mouse models of glioma. We have also demonstrated that BETi's synergize with Olaparib by downregulating expression of the BRCA-driven DNA damage repair pathway and further leverages additive effects when triply combined with other DNA damaging agents such as Lomustine to decrease tumor burden and improve survival in patient-derived mouse models of GBM and medulloblastoma. We therefore hypothesize that the synergistic and additive effects of this triple combination seen in our preclinical studies will achieve therapeutic benefits in patients with recurrent GBM.
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Shenoy, Gautam N., Maulasri Bhatta, and Richard B. Bankert. "Tumor-Associated Exosomes: A Potential Therapeutic Target for Restoring Anti-Tumor T Cell Responses in Human Tumor Microenvironments." Cells 10, no. 11 (November 13, 2021): 3155. http://dx.doi.org/10.3390/cells10113155.
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Exosomes are a subset of extracellular vesicles (EVs) that are released by cells and play a variety of physiological roles including regulation of the immune system. Exosomes are heterogeneous and present in vast numbers in tumor microenvironments. A large subset of these vesicles has been demonstrated to be immunosuppressive. In this review, we focus on the suppression of T cell function by exosomes in human tumor microenvironments. We start with a brief introduction to exosomes, with emphasis on their biogenesis, isolation and characterization. Next, we discuss the immunosuppressive effect of exosomes on T cells, reviewing in vitro studies demonstrating the role of different proteins, nucleic acids and lipids known to be associated with exosome-mediated suppression of T cell function. Here, we also discuss initial proof-of-principle studies that established the potential for rescuing T cell function by blocking or targeting exosomes. In the final section, we review different in vivo models that were utilized to study as well as target exosome-mediated immunosuppression, highlighting the Xenomimetic mouse (X-mouse) model and the Omental Tumor Xenograft (OTX) model that were featured in a recent study to evaluate the efficacy of a novel phosphatidylserine-binding molecule for targeting immunosuppressive tumor-associated exosomes.
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Glavic, Alvaro, José Luis Gómez-Skarmeta, and Roberto Mayor. "The homeoprotein Xiro1 is required for midbrain-hindbrain boundary formation." Development 129, no. 7 (April 1, 2002): 1609–21. http://dx.doi.org/10.1242/dev.129.7.1609.
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The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. We present evidence that equivalent, in addition to novel, interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. We made use of fusion proteins in which we combined Otx2 or Gbx2 homeodomains with the E1A activation domain or the EnR repressor element which were then injected into embryos. Our results show that Otx2 and Gbx2 are likely to be transcriptional repressors, and that these two proteins repress each other transcription. Our experiments show that the interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. In this study we also developed a novel in vitro assay for the study of the formation of this organizer. We show that conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreate the interactions required for the induction of the isthmic organizer. We have used this assay to determine which cells produce and which cells receive the Fgf signal. Finally, we have added a novel genetic element to this process, Xiro1, which encode another homeoprotein. We show that the Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, we show that this gene activates the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. We discuss a model where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer.
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Logan, M., H. G. Simon, and C. Tabin. "Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity." Development 125, no. 15 (August 1, 1998): 2825–35. http://dx.doi.org/10.1242/dev.125.15.2825.
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The wing and the leg of the chick, although homologous structures, have characteristic patterns of skeletal elements, muscles, tendons, featherbuds and scales. Despite recent advances in understanding the common genetic pathways patterning the wing and leg, the molecular nature of the specification of limb-type identity has remained elusive. Embryological experiments have indicated the existence of limb-specific territories in the flank. In the newt, deviation of nerves from the limb into the flank can induce ectopic limbs to form from this tissue. In the chick, Fibroblast growth factor (FGF)-soaked beads applied to the flank can induce ectopic formation of limbs from the surrounding tissue. In both cases, the type of limb that forms, either a wing/forelimb or leg/hindlimb, is dependent upon the location to which the limb-inducing signal is applied. We have isolated and characterised three candidate genes for controlling limb identity in the chick. Two T-box transcription factors, cTbx4 and cTbx5, are expressed in a restricted manner in the leg bud and wing buds, respectively. cPtx1, a member of the Otx-related subclass of paired-type homeodomain proteins, is expressed exclusively in the leg bud. Using FGF to induce ectopic limb buds of wing, leg and intermediate identity, we show that early expression of cTbx5, cTbx4 and cPtx1 in the induced limb buds correlates with later wing- or leg-type identity of ectopic limbs. We observe a general correlation between the location of an ectopic outgrowth induced by FGF and the identity of the resulting limb but, significantly, we report that there is no definitive rostral-caudal level that divides the ectopic wing and leg territories.
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Sincennes, Marie-Claude, Virginie Sanguin-Gendreau, Richard Martin, Benoit Grondin, Mathieu Tremblay, Francesco Cerisoli, Christina Magli, and Trang Hoang. "Otx1 Enhances Erythroid Differenciation through Activation of the SCL Gene." Blood 106, no. 11 (November 16, 2005): 833. http://dx.doi.org/10.1182/blood.v106.11.833.833.
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Abstract Members of the paired class of homeobox proteins are critical determinants of left-right asymmetry and establish the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that mice lacking Otx1, a bicoid homeodomain-containing gene, exhibit an impairment of the erythroid compartment, associated with decreased SCL levels. In the present study, we show that Otx1 is coexpressed with SCL in yolk sac during embryonic development; in differentiating embryonic stem cells, Otx1 is upregulated with SCL in both primitive and definitive erythroid colonies, while Otx expression is absent in cardiomyocytes and skeletomyocytes. To address the role of Otx1 in hematopoiesis, we overexpressed Otx1 in primary hematopoietic cells using the MSCV retrovirus. The gain of Otx1 function gives rise to a 6-fold increase in endogenous SCL levels together with an increase in TER119-positive erythroid cells. Strikingly, the generation of CD11b-positive myeloid cells was almost abrogated by ectopic Otx1 expression, suggesting that Otx1 favours the erythroid lineage at the expense of the myeloid lineage. Furthermore, we took several approaches to provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. Indeed, Otx1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity is dependent on the proximal GATA site of the SCL promoter. Next, we show by chromatin immunoprecipitation that Otx1 and GATA-1 occupy the SCL proximal promoter in vivo in erythroid cells. At the molecular level, we show that Otx1 physically interacts with GATA-1 in erythroid cells, and the homeodomain of Otx1 is sufficient for this interaction. Finally, a gain of function of SCL rescues the erythroid deficiency of Otx1−/− mice, consistent with the model in which SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL are involved in the same genetic pathway to specify the erythroid fate during hematopoiesis.
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Wang, Hai-He, Hai-Hui Yu, and Sek-Man Wong. "Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion." Journal of General Virology 85, no. 8 (August 1, 2004): 2447–57. http://dx.doi.org/10.1099/vir.0.80070-0.
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Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T→C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.
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Filpa, Viviana, Elisa Carpanese, Silvia Marchet, Cristina Pirrone, Andrea Conti, Alessia Rainero, Elisabetta Moro, et al. "Nitric oxide regulates homeoprotein OTX1 and OTX2 expression in the rat myenteric plexus after intestinal ischemia-reperfusion injury." American Journal of Physiology-Gastrointestinal and Liver Physiology 312, no. 4 (April 1, 2017): G374—G389. http://dx.doi.org/10.1152/ajpgi.00386.2016.
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Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2. NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.
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Song, Zhuolin, Lin Feng, Yuankui Leng, Mingzhu Huang, Hao Fang, Weipeng Tong, Xuelan Chen, and Yonghua Xiong. "Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading." Toxins 13, no. 11 (November 5, 2021): 781. http://dx.doi.org/10.3390/toxins13110781.
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Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.
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Ma, Xue, Jian Yang, Ting Liu, Jing Li, Yanyu Lan, Yonglin Wang, Aimin Wang, Ye Tian, and Yongjun Li. "Gukang Capsule Promotes Fracture Healing by Activating BMP/SMAD and Wnt/β-Catenin Signaling Pathways." Evidence-Based Complementary and Alternative Medicine 2020 (September 29, 2020): 1–12. http://dx.doi.org/10.1155/2020/7184502.
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Background. Gukang capsule (GKC) is a traditional Chinese medicine formulation which has been used extensively in the clinical treatment of bone fractures. However, the mechanisms underlying its effects on fracture healing remain unclear. Methods. In this study we used a rabbit radius fracture model, and we measured the serum content of bone alkaline phosphatase (ALP), calcium, and phosphorus and examined pathology of the fracture site as indicators of the fracture healing effects of GKC. SaOS-2 human osteosarcoma cells were used to measure (i) ALP activity, (ii) ornithine transcarbamylase (OTC), calcium, and mineralization levels, (iii) the expression of osteogenic-related genes, that is, runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen I (COL-I), osteopontin (OPN), OTC, and osterix (Osx), and (iv) the expression of key proteins in the Wnt/β-catenin and BMP/SMAD signaling pathways to study the mechanisms by which GKC promotes fracture healing. Results. We found that GKC effectively promotes radius fracture healing in rabbits and enhances ALP activity, increases OTC and calcium levels, and stimulates the formation of mineralized nodules in SaOS-2 cells. Moreover, COL-I, OTC, Osx, BMP2, and OPN expression levels were higher in SaOS-2 cells treated with GKC than control cells. GKC upregulates glycogen synthase kinase 3β (GSK3β) phosphorylation and Smad1/5 and β-catenin protein levels, thereby activating Wnt/β-catenin and BMP/Smad signaling pathways. Inhibitors of the Wnt/β-catenin and BMP/Smad signaling pathways (DKK1 and Noggin, respectively) suppress the osteogenic effects of GKC. Conclusions. GKC promotes fracture healing by activating the Wnt/β-catenin and BMP/Smad signaling pathways and increasing osteoprotegerin (OPG) secretion by osteoblasts (OBs), which prevents receptor activator of nuclear factor kappa B ligand (RANKL) binding to RANK.
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Riyahi, Niknam, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, et al. "Abstract 2017: Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2017. http://dx.doi.org/10.1158/1538-7445.am2022-2017.
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Abstract Osteosarcoma (OS) is an aggressive pediatric cancer with ~35% of patients developing metastasis over time. The survival rate for metastatic and relapsed OS patients is <30% and there is currently no standardized salvage therapy. Lack of efficacy is attributed to extensive genetic complexity present in OS that is partly due to moderate levels of replication stress (RS). While high levels of RS can induce cell death, moderate RS levels may cause genomic instability that contributes to progression of OS. Therefore, induction of RS to high levels, especially in genetically complex cancers like OS, could be a promising therapeutic strategy. Bromodomain and extra-terminal domain (BET) proteins (BRD2,3,4) are a family of epigenetic readers that not only regulate gene expression networks, but also regulate DNA replication and RS. BRD4 directly regulates major factors involved in DNA replication and checkpoint signaling. Thus, disruption of BRD4 function should exacerbate RS to levels that cause cell death. The objective of this study is to test the hypothesis that BET inhibition potentiates the efficacy of current salvage therapy through RS induction in aggressive OS. The effects of BET inhibitors (BETi), AZD5153 and OTX-015, as single agents and in combination with drugs used in salvage therapy such as topotecan were evaluated for effects on OS cell growth, PARP cleavage, and the DNA damage repair network. BET knockdown experiments were performed to evaluate target selectivity and dependency. In vivo efficacy and safety studies focused on patient-derived xenografts (PDXs) of relapsed OS. TT2-77 xenoline, Saos2, G292, and U2OS cell lines were selected for in vitro experiments. Combination index and Bliss independence analyses demonstrated additive to synergistic cell growth inhibition upon treatment with clinically relevant concentrations of BETi+topotecan. Significant increase in PARP cleavage was observed in the combination compared to single agent, indicating enhancement of apoptosis. Moreover, Western analyses demonstrated that BETi induces its effect, at least partly, via decreased CHK1 activation and increased DNA damage. Selective siRNA treatments illustrated that transient knockdown of individual BET proteins was not sufficient for potentiation of topotecan-induced cell death in OS cells, indicating that simultaneous knockdown of BETs may be required. Dose-finding studies of AZD5153 in relapsed OS PDXs that harbor replication stress signatures (TT2-77 and PDX96) indicated that daily doses of 1.25 or 2.5 mg/kg AZD5153 were well tolerated and effective in partially suppressing tumor growth compared to vehicle (p<0.05, Two-way ANOVA; Holm-Sidak). In vivo combination treatments of BETi+topotecan are in progress. These data collectively suggest that BET inhibition alongside salvage therapy holds promise as a novel treatment strategy for inducing RS-mediated cell death in aggressive OS. Citation Format: Niknam Riyahi, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathy Coy, Henry Mang, Reagan K. Wohlford, Anthony L. Sinn, Emily S. Sims, Matt J. Repass, Nuri Damayanti, Farinaz Barghi, Harlan E. Shannon, Michael J. Ferguson, Jamie L. Renbarger, Karen E. Pollok. Therapeutic induction of replication stress in the context of salvage therapy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2017.
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Johnson, D. G., L. Carayannopoulos, J. D. Capra, P. W. Tucker, and J. H. Hanke. "The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes." Molecular and Cellular Biology 10, no. 3 (March 1990): 982–90. http://dx.doi.org/10.1128/mcb.10.3.982-990.1990.
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All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.
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Johnson, D. G., L. Carayannopoulos, J. D. Capra, P. W. Tucker, and J. H. Hanke. "The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes." Molecular and Cellular Biology 10, no. 3 (March 1990): 982–90. http://dx.doi.org/10.1128/mcb.10.3.982.
Full textAbstract:
All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.
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Wang, Guangfeng, Eric Liang, Chunyang Tang, Li Rui, Ping Min, Jing Lv, Yangfeng Ge, et al. "Abstract 5439: MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5439. http://dx.doi.org/10.1158/1538-7445.am2022-5439.
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Abstract Multiple myeloma (MM) accounts for about 2% of all cancers and 18% of all hematologic malignancies in the US. Newly developed treatments (e.g., immunomodulators, proteasome inhibitors, monoclonal antibodies) have significantly improved outcomes, but MM inevitably relapses and is considered incurable. Genomic analysis shows that the TP53 gene encoding tumor suppressor protein p53 is infrequently mutated in patients with MM, of whom about 82% retain wild-type (WT) TP53. Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that inhibits p53 via proteasome degradation. Proteasome inhibitors might help to stabilize p53 and synergize with MDM2 inhibitors. Therefore, MDM2 inhibitors that activate p53 might constitute an attractive pharmacologic approach to MM. Alrizomadlin (APG-115) is an investigational, new small molecule targeting the p53/MDM2 interaction and is in clinical development for solid and hematologic cancers. This study aimed to evaluate whether alrizomadlin can potentiate the antitumor effects of proteasome inhibitors in MM. In cell-based antiproliferation studies, alrizomadlin demonstrated selective activity against WT TP53 MM cell lines, including MOLP-8, H929, and MM1S, with IC50 values of 0.495 ± 0.132 µM, 0.259 ± 0.251 µM, and 0.325 ± 0.105 µM, respectively. In contrast, alrizomadlin was inactive against TP53-mutant MM cell lines (IC50 >10 µM). In MM cell lines, APG-115 exhibited synergistic activity with anti-MM agents (e.g., carfilzomib, lenalidomide, melphalan, selinexor) and other targeted agents (e.g., panobinostat, bromodomain inhibitor OTX-015, tumor necrosis factor-related apoptotic-inducing ligand). In vivo studies showed that coadministration of alrizomadlin with proteasome inhibitor bortezomib or carfilzomib enhanced tumor regression (vs. single agents) in H929 xenograft models. As to mechanism, alrizomadlin likely disrupts MDM2/p53 interactions and induces accumulation of p53, MDM2, and downstream proteins 4 hours after treatment. The proteasome inhibitors induced dose-related increases in p53 protein expression 24 hours after treatment. The combination of carfilzomib with alrizomadlin further promoted accumulation of p53 and MDM2 and increased phosphorylation of p53. As a transcription factor, p53: (1) activated expression of p21 to cause cell cycle arrest (2) augmented downstream BCL-2-associated X protein (BAX) and p53-upregulated modulator of apoptosis (PUMA), (3) increased cleavage of caspase-3 and poly [ADP-ribose] polymerase 1 (PARP-1; hallmarks of apoptosis), and (4) increased apoptosis and associated antitumor effects. In conclusion, our results demonstrate that the combination of MDM2 inhibitor APG-115 and proteasome inhibitors have synergistic antitumor effects on MM tumors harboring WT TP53. These data lay a foundation for clinical trials of a new and novel therapeutic option for patients with refractory MM. Citation Format: Guangfeng Wang, Eric Liang, Chunyang Tang, Li Rui, Ping Min, Jing Lv, Yangfeng Ge, Fei Zhang, Lvcheng Wang, Jingjin Shang, Dajun Yang, Yifan Zhai. MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5439.
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Boi, Michela, Paola Bonetti, Maurilio Ponzoni, Maria Grazia Tibiletti, Anastasios Stathis, Esteban Cvitkovic, Giorgio Inghirami, Emanuele Zucca, and Francesco Bertoni. "The Brd-Inhibitor OTX015 Shows Pre-Clinical Activity in Anaplastic Large T-Cell Lymphoma (ALCL)." Blood 120, no. 21 (November 16, 2012): 4872. http://dx.doi.org/10.1182/blood.v120.21.4872.4872.
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Abstract Abstract 4872 Background: ALCL, is clinically/biologically heterogeneous disease, including ALK+ and ALK- systemic forms. Despite the progresses in understanding the molecular pathogenesis of ALCL, the therapy is still based on chemotherapy, thus the identification of new treatment modalities is needed. Bromodomain-containing proteins are components of transcription factors complexes and determinants of epigenetic memory. Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family, have recently shown antitumor activity in different hematological malignancies models. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a panel of ALCL cell lines. Material and Methods: Eight established human cell lines derived from ALK+ and ALK- anaplastic large cell lymphoma (ALCL) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72h exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed on using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: We assessed OTX-015 anti-proliferative activity in eight ALCL cell lines. The majority (5/8) of the cell lines were sensitive, with IC50 between 36 and 546 nM. There was no apparent difference between ALK+(6) and ALK- (2) cell lines. Cell cycle analyses revealed G1 arrest and a concomitant decrease of the S phase after 24h OTX015 exposure in 4/4 ALCL cell lines, without an increase in cell death, suggesting a cytostatic effect of OTX015. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in the most sensitive ALK+ALCL cell line. To understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after treatment. We observed that OTX015 suppressed the transcription of MYCgene and some of its downstream target genes (such as NCL and CAD) in 4/4 ALCL cell lines, with less efficacy in the most resistant one. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several ALCL cell lines. The down-regulation of MYC gene, followed by cell cycle G1 arrest and increase of cellular senescence, was observed after OTX015 treatment, appearing one of the possible mechanisms of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in ALCL and in other mature T-cell tumors. Disclosures: Bonetti: OncoEthix SA: Research Funding. Cvitkovic:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Inghirami:OncoEthix SA: Research Funding. Bertoni:OncoEthix SA: Research Funding.
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Gautron, Joël, Sophie Réhault-Godbert, Géraldine Pascal, Yves Nys, and Maxwell T. Hincke. "Ovocalyxin-36 and other LBP/BPI/PLUNC-like proteins as molecular actors of the mechanisms of the avian egg natural defences." Biochemical Society Transactions 39, no. 4 (July 20, 2011): 971–76. http://dx.doi.org/10.1042/bst0390971.
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The chicken egg possesses physical and chemical barriers to protect the embryo from pathogens. OCX-36 (ovocalyxin-36) was suggested to be a 36 kDa eggshell-specific protein that is secreted by the regions of the oviduct responsible for eggshell formation. Its expression is strongly up-regulated during shell calcification. This protein was also detected in vitelline membrane and expressed in gut tissues. Analysis of the OCX-36 protein sequence revealed that OCX-36 is related to the BPI (bactericidal permeability-increasing proteins)/LBP [LPS (lipopolysaccharide)-binding proteins]/PLUNC (palate, lung and nasal epithelium clone) superfamily, and that there are strong similarities between the exon/intron organization of the mammalian LBP/BPI and the avian OCX-36 genes. A recent study revealed that OCX-36 originates from a tandem duplication of an ancestral BPI/LBP/PLUNC gene, after the divergence of birds and mammals. Its antimicrobial activity was recently investigated and it was shown that OCX-36 binds to LPS from Escherichia coli. High-throughput methodologies have led to the identification of approximately 1000 new egg proteins. Among these are LBP/BPI proteins that might play a role in the natural defences of the egg to protect the embryo during its development in the external milieu, and may function to keep the table egg free of pathogens. The function of these BPI-like molecules is the subject of intense research to characterize their putative LPS-binding properties and antimicrobial activity.
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Barbuto, Richard, and Jane Mitchell. "Regulation of the osterix (Osx, Sp7) promoter by osterix and its inhibition by parathyroid hormone." Journal of Molecular Endocrinology 51, no. 1 (May 16, 2013): 99–108. http://dx.doi.org/10.1530/jme-12-0251.
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Osterix (Osx, Sp7) is a zinc-finger transcription factor belonging to the specificity protein (Sp) family expressed in cells of the osteoblast lineage in the developing skeleton where it regulates expression of a number of osteoblastic genes. We previously reported inhibition of osterix mRNA and protein by parathyroid hormone (PTH) stimulation of cAMP in osteoblasts. We here show that Osx expression in osteoblasts is regulated by Sp proteins as demonstrated by mithramycin A inhibition of Osx mRNA and OSX protein levels. Mutation of putative transcription factor binding sites within the Osx promoter demonstrated a tandem repeat sequence that selectively binds OSX but not other Sp factors expressed in osteoblasts (Sp1, Sp3, or Tieg (Klf10)). Mutation of either or both the repeat sequences inhibited 90% of the promoter activity and also abrogated some of the PTH-mediated inhibition of the promoter. Previous studies have shown growth factor regulation of Osx expression by MAPK proteins, particularly p38 phosphorylation of OSX that increases its transcriptional activity. PTH stimulation of osteoblasts inhibits MAPK components (ERK, JNK, and p38) but inhibition of Osx mRNA and protein expression by PTH was selectively mimicked by p38 inhibition and expression of constitutively active MKK6, which stimulates p38, blocked PTH inhibition of OSX. Together, our studies suggest that OSX autoregulation is a major mechanism in osteoblasts and that PTH stimulation inhibits osterix by inhibition of p38 MAPK regulation of OSX.
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Cuthbertson, Leslie, Iain L. Mainprize, James H. Naismith, and Chris Whitfield. "Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria." Microbiology and Molecular Biology Reviews 73, no. 1 (March 2009): 155–77. http://dx.doi.org/10.1128/mmbr.00024-08.
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SUMMARY Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology.
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Xing, Weirong, Sheila Pourteymoor, and Subburaman Mohan. "Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway." Physiological Genomics 43, no. 12 (June 2011): 749–57. http://dx.doi.org/10.1152/physiolgenomics.00229.2010.
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Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs.
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Xu, Weixiang, Mingyang Wang, Gengyuan Cui, Lin Li, Danyang Jiao, Beibei Yao, Ketao Xu, et al. "Astaxanthin Protects OTA-Induced Lung Injury in Mice through the Nrf2/NF-κB Pathway." Toxins 11, no. 9 (September 17, 2019): 540. http://dx.doi.org/10.3390/toxins11090540.
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The aim of this research was to evaluate the potential protective mechanism of astaxanthin (ASTA) against oxidative damage and inflammation caused by ochratoxin (OTA) in mouse lung. We divided mice into a control group (CG), an OTA group (PG), an astaxanthin group (AG), and an OTA+ASTA group (JG). Oxidative indices (malondialdehyde (MDA), total superoxide dismutase (T-SOD), and reduced glutathione (GSH)) and inflammatory markers (interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α)) were assayed in the lung, and the lung-weight-to-body-weight ratio was calculated. Apoptosis was detected in pathological sections by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Oxidative damage and inflammation were detected in the lung of mice after exposure to OTA. Besides, Nrf2- and NF-κB-pathway-associated proteins were detected by Western blot. In contrast with OTA, ASTA significantly raised the expression of Nrf2, HO-1, and MnSOD, while the expression of other proteins (Keap1, TLR4, and NF-κB) was significantly decreased. These results indicate that ASTA exerted protective effects against OTA-induced oxidative damage and inflammation in the lung by regulating the Nrf2 and NF-κB pathways.
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Gomez, Marcus, Vijay G. Ramakrishnan, Vivek Prasad, Teresa K. Kimlinger, Utkarsh Painuly, Lintao Bi, Vincent Rajkumar, and Shaji Kumar. "Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins." Blood 124, no. 21 (December 6, 2014): 2088. http://dx.doi.org/10.1182/blood.v124.21.2088.2088.
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Abstract Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and a Results: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX. Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. Disclosures No relevant conflicts of interest to declare.
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Schwerdt, Gerald, Michael Kopf, and Michael Gekle. "The Impact of the Nephrotoxin Ochratoxin A on Human Renal Cells Studied by a Novel Co-Culture Model Is Influenced by the Presence of Fibroblasts." Toxins 13, no. 3 (March 18, 2021): 219. http://dx.doi.org/10.3390/toxins13030219.
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The kidney is threatened by a lot of potentially toxic substances. To study the influence of the nephrotoxin ochratoxin A (OTA) we established a cell co-culture model consisting of human renal proximal tubule cells and fibroblasts. We studied the effect of OTA on cell survival, the expression of genes and/or proteins related to cell death, extracellular matrix and energy homeostasis. OTA-induced necrosis was enhanced in both cell types in the presence of the respective other cell type, whereas OTA-induced apoptosis was independent therefrom. In fibroblasts, but not in tubule cells, a co-culture effect was visible concerning the expression of the cell-cycle-related protein p21. The expression of the epithelial-to-mesenchymal transition-indicating protein vimentin was independent from the culture-condition. The expression of the OTA-induced lncRNA WISP1-AS1 was enhanced in co-culture. OTA exposure led to alterations in the expression of genes related to energy metabolism with a glucose-mobilizing effect and a reduced expression of mitochondrial proteins. Together we demonstrate that the reaction of cells can be different in the presence of cells which naturally are close-by, thus enabling a cellular cross-talk. Therefore, to evaluate the toxicity of a substance, it would be an advantage to consider the use of co-cultures instead of mono-cultures.
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Hussein, T. S., D. A. Froiland, J. G. Thompson, and R. B. Gilchrist. "232. Oocytes prevent bovine cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins." Reproduction, Fertility and Development 17, no. 9 (2005): 91. http://dx.doi.org/10.1071/srb05abs232.
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Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell (CC) functions. Previously we have shown that the low incidence of apoptosis in CCs is due to unidentified oocyte-secreted factors (OSF) acting in an anti-apoptotic manner. Here we examine the nature of the paracrine network of oocyte BMP growth factors and their binding proteins regulating CC apoptosis. Bovine cumulus–oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytes microsurgically removed to create oocytectomized (OOX) complexes. OOX were treated with denuded oocytes (DO) or various growth factors for 24 h, then CC apoptosis was assessed using TUNEL together with confocal microscopy plus image analysis and by Western blotting for Bcl-2 and Bax. CC apoptosis was significantly (P < 0.001) reduced by DO, bone morphogenetic protein 15 (BMP15), BMP6 or BMP7 as assessed by TUNEL. Accordingly, expression of anti-apoptotic Bcl-2 was high in OOX+DO and OOX+BMP15, and low with OOX+GDF9 and OOX alone, whereas the reverse was observed for pro-apoptotic Bax. Combined treatment of OOXs with BMP6 and BMP15 did not further decrease apoptosis levels beyond that of BMP15 alone (P > 0.05), suggesting no additive effect of these two BMPs. Follistatin (FS) effectively antagonized BMP15 anti-apoptotic effects, and likewise, a BMP6 neutralizing antibody (NAb) antagonized the inhibitory effect of BMP6. Gremlin blocked BMP7 anti-apoptotic effects on CCs, but had no significant effect on BMP15. FS or BMP6. NAb antagonized ~50% of the anti-apoptotic activity of oocytes; however, these effects were not additive suggesting the additional involvement of other OSF. These results indicate for the first time that OSF (BMP15 and BMP6 in particular) maintains the low incidence of CC apoptosis by establishing a localized morphogenic gradient of bone morphogenetic proteins.
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Ferrara, Massimo, Antonia Gallo, Carla Cervini, Lucia Gambacorta, Michele Solfrizzo, Scott E. Baker, and Giancarlo Perrone. "Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius." Toxins 13, no. 12 (December 13, 2021): 892. http://dx.doi.org/10.3390/toxins13120892.
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Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.
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Tong, Weipeng, Hanpeng Xiong, Hao Fang, Yuhao Wu, Haichuan Li, Xiaolin Huang, Yuankui Leng, and Yonghua Xiong. "Bifunctional M13 Phage as Enzyme Container for The Reinforced Colorimetric–Photothermal Dual-Modal Sensing of Ochratoxin A." Toxins 15, no. 1 (December 20, 2022): 5. http://dx.doi.org/10.3390/toxins15010005.
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“Point of care” (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL−1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL−1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.
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Tao, Yue, Minhao Wu, Xing Zhou, Wu Yin, Bin Hu, Benoit de Crombrugghe, Krishna M. Sinha, and Jianye Zang. "Structural Insights into Histone Demethylase NO66 in Interaction with Osteoblast-specific Transcription Factor Osterix and Gene Repression." Journal of Biological Chemistry 288, no. 23 (April 24, 2013): 16430–37. http://dx.doi.org/10.1074/jbc.m112.446849.
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Osterix (Osx) is an osteoblast-specific transcriptional factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory factors during gene expression.
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Wu, Minhao, Yue Tao, Xing Zhou, Krishna Sinha, and Jianye Zang. "Structural Basis for Interaction Between NO66 and Osterix." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C313. http://dx.doi.org/10.1107/s2053273314096867.
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The differentiation of mesenchymal stem cells to osteoblasts is one of the critical steps of bone formation. Osterix (Osx) is an osteoblast-specific transcriptional factor required for bone formation and osteoblast differentiation, which has been shown to interact with other factors to control the expression of osteoblast-specific genes. A novel JmjC domain containing protein NO66 has been identified in the regulation network of Osx, which plays an important role in osteoblast differentiation through interaction with Osx. Here we report the crystal structure of NO66, showing it exists as a functional tetramer form. A hinge domain links N-terminal JmjC domain and C-terminal wHTH domain of NO66 and is essential for its tetrameric assembly. The oligomerization interface of NO66 provides the binding site for Osx, which interacts with a conserved fragment of Osx. Further work demonstrates that the hinge domain-dependent oligomerization is essential for NO66 to interact with Osx and controls Osx-dependent gene expression. Our finding reveals that homo-oligomerization of JmjC domain containing proteins plays a critical role in interaction with regulatory factors and may have significant physiological roles.
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Alizadeh, A., P. Akbari, S. Varasteh, S. Braber, H. Malekinejad, and J. Fink-Gremmels. "Ochratoxin A challenges the intestinal epithelial cell integrity: results obtained in model experiments with Caco-2 cells." World Mycotoxin Journal 12, no. 4 (December 4, 2019): 399–407. http://dx.doi.org/10.3920/wmj2019.2451.
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Contamination of human and animal diets with different mycotoxins have gained significant attention over the past decade. The intestinal barrier is the first site of exposure and a primary target for nutritional contaminants and hazardous substances including mycotoxins. In this study, the potential impact of ochratoxin A (OTA) on intestinal barrier integrity was highlighted using a human intestinal Caco-2 cell line. Cell viability following OTA exposure was determined by lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, markers of barrier integrity, such as transepithelial electrical resistance (TEER) as well as the permeability of Lucifer Yellow (LY) and fluorescein isothiocyanate (FITC)-dextran, were assessed. Furthermore, the protein expression of different tight junction (TJ) proteins, as main constituents of barrier integrity, was evaluated by Western blot. Results show that OTA reduces TEER values in a concentration- and time-dependent manner and increase the permeability of LY through the intestinal epithelial layer, while the cell viability did not change significantly. However, the damage was not severe enough to change the permeability to larger molecules, such as FITC-dextran. OTA exposure down-regulated the expression of TJ proteins claudin-1, -3 and -4 and up-regulated the expression of zona occludens 1. The observation that OTA can disrupt the epithelial barrier is of clinical importance as it may lead to an increased passage of luminal antigens into the systemic circulation.
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Parr, Jacqueline M., and Rebecca L. Remillard. "Common Confounders of Dietary Elimination Trials Contain the Antigens Soy, Pork, and Beef." Journal of the American Animal Hospital Association 50, no. 5 (September 1, 2014): 298–304. http://dx.doi.org/10.5326/jaaha-ms-6104.
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Nutritionists and dermatologists recommend avoiding flavored over-the-counter (OTC) products and medications during dietary elimination trials because those products are thought to contain common proteins that may confound the trial. The objective of this study was to determine if there are soy, pork, and beef antigens in flavored OTC products and medications and, if so, could those antigens be identified. Seven products, three OTC products and four veterinary therapeutics, were tested using enzyme-linked immunosorbent assays (ELISA) for the presence of soy, pork, and beef antigens, in addition to positive and negative controls. All OTC test products produced ELISA results in agreement with their ingredient lists. ELISA testing of veterinary therapeutic products did not agree with either their ingredient lists or product inserts because of other ingredients not listed. Veterinarians should contact manufacturers of oral therapeutics prior to prescribing them to determine other ingredients. Likewise, manufacturers should be contacted regarding “natural and artificial flavors.” Lastly, gelatin capsules may contain either beef or pork proteins and should not be administered during a trial. In conclusion, flavored medications contain the common antigens soy, pork, and beef although they may or may not be listed on the ingredient list or product insert.
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Di Cerbo, Alessandro, Antonio Scarano, Federica Pezzuto, Gianandrea Guidetti, Sergio Canello, Diego Pinetti, Filippo Genovese, and Lorenzo Corsi. "Oxytetracycline-Protein Complex: The Dark Side of Pet Food." Open Public Health Journal 11, no. 1 (April 30, 2018): 162–69. http://dx.doi.org/10.2174/1874944501811010162.
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Background:Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and medicalization representing another way of antibiotic resistance transmission.Objective:We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and Oxytetracycline (OTC), an approved antibiotic for use in zootechny, which might determine a toxic effect on K562 cells.Method:Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens according to standard withdrawal times and from untreated chickens at 24, 48 and 72h of incubation.Results:Cell culture medium with ground bone from chickens reared in the presence of OTC (OTC-CCM) resulted significantly cytotoxic at every incubation time regardless of the bone concentration while cell culture medium with ground bone from chickens reared without OTC (BIO-CCM) resulted significantly cytotoxic only after 72h of incubation. HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium until 1 μg/mL.Conclusion:The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of incubation.
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Emch, Andrew J., and Jason J. Nichols. "Proteins Identified From Care Solution Extractions of Silicone Hydrogels." Optometry and Vision Science 86, no. 2 (February 2009): E123—E131. http://dx.doi.org/10.1097/opx.0b013e318194eb01.
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Khatoon, A., M. Z. Khan, Z. Abidin, A. Khan, and M. K. Saleemi. "Mitigation potential of distillery sludge against ochratoxin A induced immunological alterations in broiler chicks." World Mycotoxin Journal 10, no. 3 (September 7, 2017): 255–62. http://dx.doi.org/10.3920/wmj2016.2159.
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Ochratoxin A (OTA) is a potent mycotoxin injurious to poultry health and an alarming factor for poultry industry while distillery sludge (DS) is a waste product of molasses based industries rich in proteins and certain essential vitamins and other nutrients. The present study was done to estimate the immunological alterations induced by OTA in broiler chicks and amelioration of these alterations by dietary supplementation of DS. For this purpose, 480 one-day old broiler chicks procured from a local hatchery, were divided into sixteen equal groups and were given different combinations of OTA (150, 300 and 1000 µg/kg feed) and DS (5, 10 and 20 g/kg feed). Parameters studied were antibodies response to sheep red blood cells (SRBCs), lymphoproliferative response to PHA-P and phagocytic index as studied by carbon clearance assay. The results of this study showed that feeding DS with 150 and 300 µg/kg OTA ameliorated OTA induced alterations, but this amelioration was partial when 1000 µg/kg OTA was used along with DS. From this study it could be concluded that DS has beneficial effects in birds suffering from ochratoxicosis. However, the proper level of DS to produce such mitigation against specific level of OTA is yet to be determined.
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Kutoh, E., P. E. Strömstedt, and L. Poellinger. "Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1." Molecular and Cellular Biology 12, no. 11 (November 1992): 4960–69. http://dx.doi.org/10.1128/mcb.12.11.4960-4969.1992.
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The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.
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Kutoh, E., P. E. Strömstedt, and L. Poellinger. "Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1." Molecular and Cellular Biology 12, no. 11 (November 1992): 4960–69. http://dx.doi.org/10.1128/mcb.12.11.4960.
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The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.
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Terada, Atsushi, Takashi Honda, Hideo Fukuhara, Kazumasa Hada, and Makoto Kimura. "Characterization of the Archaeal Ribonuclease P Proteins from Pyrococcus horikoshii OT3." Journal of Biochemistry 140, no. 2 (August 1, 2006): 293–98. http://dx.doi.org/10.1093/jb/mvj144.
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Sztul, E. S., T. W. Chu, A. W. Strauss, and L. E. Rosenberg. "Translocation of precursor proteins into the mitochondrial matrix occurs through an environment accessible to aqueous perturbants." Journal of Cell Science 94, no. 4 (December 1, 1989): 695–701. http://dx.doi.org/10.1242/jcs.94.4.695.
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We have identified translocational intermediates generated during import of precursor proteins into the mitochondrial matrix and have characterized their association with mitochondrial membranes. Partially translocated forms of mitochondrial malate dehydrogenase (MDH) and ornithine transcarbamylase (OTC) were generated during import of the corresponding precursors (pMDH and pOTC) into mitochondria at 2 degrees C. Import at this temperature results in the formation of intermediate-sized MDH (iMDH) and OTC (iOTC) produced by the removal of a portion of the leader peptide, and in the production of mature-sized MDH. All of these forms contain NH2 termini located within the mitochondrial matrix, although the majority of their polypeptide chains remain extramitochondrial. All three are strongly associated with mitochondrial membranes, but can be extracted by protein denaturants such as urea. These translocational intermediates appear to be hydrophilic proteins, on the basis of their partitioning properties during extraction with the nonionic detergent Triton X-114. The data indicate that the translocation of polypeptide chains into mitochondria occurs in a microenvironment that is aqueous in nature and is mediated by integral membrane proteins.