Identification of clinically approved small molecules that inhibit growth and affect transcript levels of developmentally regulated genes in the African trypanosome

Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. However, the majority of cells that undergo reprogramming become trapped in a partially reprogrammed state that is characterised by the down-regulation of somatic cell marker genes, incomplete reactivation of pluripotency genes, maintenance of viral expression, and the inability to form chimeras. Several small molecules, which act on specific signaling pathways or chromatin modifications, have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the roadblocks encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and reactivate the embryonic stem cell genome. Chemical treatment of cells before reprogramming can remodel the epigenetic landscape to be more like that of embryonic stem cell by removing the repressive epigenetic marks and relaxing chromatin structure to allow the reprogramming factors easier access to target genes. In the present study, we assessed the effect of pretreatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen-activated protein kinase inhibitor; CHIR99021, a glycogen synthase kinase-3 inhibitor; and NuP0178 (NuPotential, Baton Rouge, LA, USA), a G9a histone methyltransferase inhibitor. Cells were seeded at a density of 0.7 × 106 and expanded before being divided evenly into 2 groups. Control cells were cultured in complete culture medium (DMEM with 10% fetal bovine serum), whereas treatment cells were cultured in complete culture medium containing 0.5 mM PD0325901, 3 mM CHIR99021, and 1.8 mM NuP0178. Messenger RNA was isolated from cell cultures using Dynabeads® mRNA DIRECT™ Kit (Dynal Inc., Lake Success, NY, USA) on Day 7 and 14, and the resulting RNA products were transcribed into cDNA using Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative RT–PCR was performed to measure transcript levels of Oct-4, Nanog, Sox-2, and poly adenylate polymerase in treated and untreated cells at Days 7 and 14. Transcript levels were quantified by relative quantification using the ΔΔCt method and expressed as ratios of target genes (Oct-4, Nanog, Sox-2) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine fetal fibroblast cells. No difference in expression levels between untreated and treated cells was detected at either Day 7 or 14. Currently, we are utilising chromatin immunoprecipitation to examine chromatin and DNA methylation patterns around the promoters of Oct-4, Nanog, and Sox-2 to further elucidate the effects of treatment with this combination of small molecule inhibitors.

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Toxoplasma gondii Asexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression

Eukaryotic Cell ◽

10.1128/ec.1.3.329-340.2002 ◽

2002 ◽

Vol 1 (3) ◽

pp. 329-340 ◽

Cited By ~ 136

Author(s):

Michael D. Cleary ◽

Upinder Singh ◽

Ira J. Blader ◽

Jeremy L. Brewer ◽

John C. Boothroyd

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Expression Profiles ◽

Protozoan Parasite ◽

Transcript Abundance ◽

Surface Proteins ◽

Asexual Development ◽

Mrna Levels ◽

Developmentally Regulated ◽

Transcript Levels

ABSTRACT Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of ∼4,400 Toxoplasma cDNAs, representing a minimum of ∼600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

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Coordinate regulation of SR Ca(2+)-ATPase and phospholamban expression in developing murine heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.272.1.h57 ◽

1997 ◽

Vol 272 (1) ◽

pp. H57-H66 ◽

Cited By ~ 5

Author(s):

J. M. Harrer ◽

K. Haghighi ◽

H. W. Kim ◽

D. G. Ferguson ◽

E. G. Kranias

Keyword(s):

Important Determinant ◽

Sr Proteins ◽

Stoichiometric Ratio ◽

Wild Type ◽

Developmentally Regulated ◽

Cardiac Sarcoplasmic Reticulum ◽

Transcript Levels ◽

Coordinate Regulation ◽

Biochemical Studies ◽

Pump Activity

Phospholamban, the regulator of Ca(2+)-adenosinetriphosphatase (ATPase) activity in cardiac sarcoplasmic reticulum (SR), is an important determinant of basal myocardial performance. To determine whether phospholamban expression is developmentally regulated in the mouse and whether such regulation reflects alterations in Ca2+ pump activity, hearts from different stages of development were processed for molecular biological and biochemical studies. Both phospholamban and Ca(2+)-ATPase mRNAs were approximately 40% of adult (100%) levels at birth and gradually increased to approach adult levels by day 15 of development. These changes in transcript levels were indicative of changes at the protein level for both phospholamban and Ca(2+)-ATPase. Analysis of the initial rates of Ca2+ uptake demonstrated that over the course of development the upregulation of Ca(2+)-ATPase correlated with increases in the maximal rates of Ca2+ uptake and the constant apparent stoichiometric ratio of phospholamban to Ca(2+)-ATPase correlated with maintenance of a constant affinity of this enzyme for Ca2+ (0.25 +/- 0.03 microM Ca2+). Furthermore, targeted ablation of phospholamban in the mouse resulted in a much higher affinity of Ca2+ uptake for Ca2+ (0.10 +/- 0.02 microM Ca2+) than that observed in wild-type hearts, and this increased affinity was also maintained across different stages of postnatal development. These findings suggest that phospholamban is a major regulator of the affinity of Ca(2+)-ATPase for Ca2+, and coordinate regulation of the expression levels of these two SR proteins may be necessary for maintaining Ca2+ homeostasis in the developing mammalian heart.

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The DNA damage response is developmentally regulated in the African trypanosome

DNA Repair ◽

10.1016/j.dnarep.2018.11.005 ◽

2019 ◽

Vol 73 ◽

pp. 78-90 ◽

Cited By ~ 4

Author(s):

J.P. Vieira-da-Rocha ◽

D.G. Passos-Silva ◽

I.C. Mendes ◽

E.A. Rocha ◽

D.A. Gomes ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Developmentally Regulated ◽

African Trypanosome ◽

Damage Response

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Small-Molecule Screens Reveal Novel Haustorium Inhibitors in the Root Parasitic PlantTriphysaria versicolor

Phytopathology ◽

10.1094/phyto-04-19-0115-r ◽

2019 ◽

Vol 109 (11) ◽

pp. 1878-1887 ◽

Cited By ~ 3

Author(s):

Yaxin Wang ◽

Daniel Steele ◽

Maylin Murdock ◽

Seigmund Lai ◽

John Yoder

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Early Stage ◽

Structural Analog ◽

Parasitic Plant ◽

In Vitro Assay ◽

Vitro Assay ◽

Transcript Levels ◽

Parasitic Weeds

Root parasitic weeds in Orobanchaceae pose a tremendous threat to agriculture worldwide. We used an in vitro assay to screen libraries of small molecules for those capable of inhibiting or enhancing haustorium development in the parasitic plant Triphysaria versicolor. Several redox-modifying molecules and one structural analog of 2,6-dimethoxybenzoquine (DMBQ) inhibited haustorium development in the presence of the haustorium-inducing factor DMBQ, some of these without apparent growth inhibition to the root. Triphysaria seedlings were able to acclimate to some of these redox inhibitors. Transcript levels of four early-stage haustorium genes were differentially influenced by inhibitors. These novel haustorium inhibitors highlight the importance of redox cycling for haustorium development and suggest the potential of controlling parasitic weeds by interrupting early-stage redox-signaling pathways.

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Vacuolar processing enzymes, AmVPE1 and AmVPE2, as potential executors of ethylene regulated programmed cell death in the lace plant (Aponogeton madagascariensis)

Botany ◽

10.1139/cjb-2017-0184 ◽

2018 ◽

Vol 96 (4) ◽

pp. 235-247 ◽

Cited By ~ 4

Author(s):

Gaolathe Rantong ◽

Arunika H.L.A.N. Gunawardena

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Leaf Development ◽

Cell Types ◽

Ethylene Biosynthesis ◽

Activity Levels ◽

Developmentally Regulated ◽

Transcript Levels ◽

Processing Enzymes ◽

Laser Capture Microscopy

Perforation formation in Aponogeton madagascariensis (Mirb.) H.Bruggen (lace plant) is an excellent model for studying developmentally regulated programmed cell death (PCD). In this study, we isolated and identified two lace plant vacuolar processing enzymes (VPEs) and investigated their involvement in PCD and throughout leaf development. Lace plant VPE transcript levels were determined during seven different stages of leaf development. PCD and non-PCD cells from “window” stage leaves (in which perforations are forming) were separated through laser-capture microscopy and their transcript levels were also determined. VPE activity was also studied between the cell types, through a VPE activity-based probe JOPD1. Additionally, VPE transcript levels were studied in plants treated with an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG). The two isolated VPEs, AmVPE1 and AmVPE2, are vegetative type VPEs. AmVPE1 had higher transcript levels during a pre-perforation developmental stage, immediately prior to visible signs of PCD. AmVPE2 transcript levels were higher later during window and late window stages. Both VPEs had higher transcript and activity levels in PCD compared with the non-PCD cells. AVG treatment inhibited PCD and associated increases in VPE transcript levels. Our results suggested that VPEs are involved in the execution of the ethylene-related PCD in the lace plant.

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The African trypanosome cyclophilin A homologue contains unusual conserved central and N-terminal domains and is developmentally regulated

Gene ◽

10.1016/s0378-1119(02)00559-0 ◽

2002 ◽

Vol 290 (1-2) ◽

pp. 181-191 ◽

Cited By ~ 12

Author(s):

Roger Pellé ◽

Francis McOdimba ◽

Francis Chuma ◽

Delia Wasawo ◽

Terry W Pearson ◽

...

Keyword(s):

Cyclophilin A ◽

Developmentally Regulated ◽

African Trypanosome ◽

Terminal Domains

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Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors

Journal of Cellular and Molecular Medicine ◽

10.1111/j.1582-4934.2011.01482.x ◽

2012 ◽

Vol 16 (8) ◽

pp. 1866-1877 ◽

Cited By ~ 14

Author(s):

Rutger J. Wierda ◽

Hedwich F. Kuipers ◽

Marja C. J. A. van Eggermond ◽

Anne Benard ◽

Jan C. van Leeuwen ◽

...

Keyword(s):

Small Molecules ◽

Immune Cells ◽

Transcript Levels ◽

Epigenetic Control

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Activation of the autophagy pathway decreases dengue virus infection in Aedes aegypti cells

Parasites & Vectors ◽

10.1186/s13071-021-05066-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Tse-Yu Chen ◽

Chelsea T. Smartt

Keyword(s):

Aedes Aegypti ◽

Dengue Virus ◽

Small Molecules ◽

Control Strategies ◽

Virus Titer ◽

Denv Infection ◽

Mosquito Vector ◽

Transcript Levels ◽

Rapamycin Treatment ◽

Autophagy Pathway

Abstract Background Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50–100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito. Methods To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway. Results At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells. Conclusions Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito’s immune response against DENV. Graphical Abstract

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Dietary Supplementation with Ipriflavone Decreases Liver Iron Loading in Wild Type Mice

Blood ◽

10.1182/blood.v124.21.4025.4025 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4025-4025

Author(s):

Bonnie Patchen ◽

Vera Gaun ◽

Aaron Cheng ◽

Paula G. Fraenkel

Keyword(s):

Iron Overload ◽

Small Molecules ◽

Iron Content ◽

Iron Homeostasis ◽

Conflicts Of Interest ◽

Iron Release ◽

Liver Iron ◽

Transcript Levels ◽

Liver Iron Content ◽

Significant Difference

Abstract Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release by causing internalization and degradation of the iron exporter ferroportin. Because its levels are inappropriately low in patients with iron overload syndromes, Hepcidin is a potential drug target. We previously conducted a chemical screen in human hepatocytes (HepG2 cells) to identify small molecules that upregulate Hepcidin transcript levels. One of the small molecules that we identified was ipriflavone, a synthetic isoflavone, which has been used to treat osteoporosis in postmenopausal women. To evaluate ipriflavone’s effect on iron homeostasis in a mammalian model, we placed groups of 5-week old C57BL/6 male mice on a soy-free, iron-sufficient diet, AIN-93G containing 220 mg/kg iron and 0, 250, 500 or 750 mg ipriflavone per kg of food (n=4 or 5 per group) for 50 days, then sacrificed the animals for analysis of changes in gene expression by quantitative realtime RT-PCR, liver and spleen iron content, blood indices, and intestinal ferroportin expression. While producing less than a two-fold increase in liver hepcidin transcript levels, ipriflavone supplementation was associated with a significant decrease in liver iron content (mean±SE): 39.22±2.06 µg iron/g tissue, p=0.0033, and 44.10±2.58 µg iron/g tissue, p=0.0124, at 500 and 750 mg ipriflavone per kg of food, respectively, vs 57.77±3.39 µg iron/g tissue in mice that did not receive ipriflavone. Ferroportin expression detected in intestinal epithelial cells by immunohistochemistry was notably decreased in mice receiving ipriflavone: 66.6% and 80% of the animals receiving 500 and 750 mg/kg groups, respectively, exhibited decreased ferroportin staining versus 40% of the group not receiving ipriflavone. There was no significant difference in hemoglobin, hematocrit, or spleen iron among the groups. In conclusion, we have shown that orally administered ipriflavone is effective in decreasing liver iron content and intestinal ferroportin expression in vivo. Future experiments will evaluate ipriflavone’s effects on iron homeostasis and hematopoiesis in genetic models of iron overload disorders. Disclosures No relevant conflicts of interest to declare.

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