scholarly journals Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Revathi Raman ◽  
Weam Fallatah ◽  
Ayah Al Qaryoute ◽  
Sanchi Dhinoja ◽  
Pudur Jagadeeswaran
Keyword(s):  
Factor Viia ◽  
Initial Step ◽  
Regulatory Proteins ◽  
Coagulation Cascade ◽  
Mrna Levels ◽  
Chromatin Binding ◽  
Antithrombotic Drug ◽  
Individual Gene ◽  
Laser Injury ◽  
Genome Wide

AbstractTissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

scholarly journals Management of the Bleeding Patient Receiving New Oral Anticoagulants: A Role for Prothrombin Complex Concentrates

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Lisa M. Baumann Kreuziger ◽  
Joseph C. Keenan ◽  
Colleen T. Morton ◽  
David J. Dries
Keyword(s):  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Oral Anticoagulants ◽  
New Oral Anticoagulants ◽  
Coagulation Cascade ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Clotting System ◽  
Clinical Trial Evidence

Ease of dosing and simplicity of monitoring make new oral anticoagulants an attractive therapy in a growing range of clinical conditions. However, newer oral anticoagulants interact with the coagulation cascade in different ways than traditional warfarin therapy. Replacement of clotting factors will not reverse the effects of dabigatran, rivaroxaban, or apixaban. Currently, antidotes for these drugs are not widely available. Fortunately, withholding the anticoagulant and dialysis are freqnently effective treatments, particularly with rivaroxaban and dabigatran. Emergent bleeding, however, requires utilization of Prothrombin Complex Concentrates (PCCs). PCCs, in addition to recombinant factor VIIa, are used to activate the clotting system to reverse the effects of the new oral anticoagulants. In cases of refractory or emergent bleeding, the recommended factor concentrate in our protocols differs between the new oral anticoagulants. In patients taking dabigatran, we administer an activated PCC (aPCC) [FELBA] due to reported benefit in human in vitro studies. Based on human clinical trial evidence, the 4-factor PCC (Kcentra) is suggested for patients with refractory rivaroxaban- or apixaban-associated hemorrhage. If bleeding continues, recombinant factor VIIa may be employed. With all of these new procoagulant agents, the risk of thrombosis associated with administration of factor concentrates must be weighed against the relative risk of hemorrhage.

scholarly journals Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob)

1991 ◽  
Vol 266 (24) ◽  
pp. 15949-15955
Author(s):  
T.W. Gettys ◽  
V. Ramkumar ◽  
R.J. Uhing ◽  
L. Seger ◽  
I.L. Taylor
Keyword(s):  
Regulatory Proteins ◽  
Mrna Levels ◽  
Obese Mice ◽  
Gtp Binding ◽  
And Function

scholarly journals Cellular localization and trafficking of tissue factor

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4746-4753 ◽  
Author(s):  
Samir K. Mandal ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Cellular Localization ◽  
Factor Viia ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Cell Surfaces ◽  
Caveolin 1 ◽  
Intracellular Compartments ◽  
Resultant Increase

AbstractTissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

Abstract 315: Efficacy and Safety of Rivaroxaban in in vitro Experiments With the Endothelial Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Kaname Seki ◽  
Yosuke Mizuno ◽  
Toku Sakashita ◽  
Jun Tanno ◽  
Shintaro Nakano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Umbilical Vein ◽  
Endothelial Damage ◽  
Coagulation Cascade ◽  
Elisa Assay ◽  
Mrna Levels ◽  
Factor X ◽  
Clotting Factors ◽  
Inflammatory Phase

Aim: Activated factor X (FXa) plays important roles in the thrombin generation and in inflammation, which is evoked during the endothelial damage. Although rivaroxaban is a selective FXa antagonist, it is one of the key therapies in ischemic heart disease, and yet its function in the state of inactivated coagulation cascade is uncertain. Rivaroxaban blocks FXa in the blood but not the tissue, while factor X is converted to FXa only when glutamic acid is changed to γ-carboxyglutamic acid by vitamin K following the intrinsic clotting factors and/or cellular injury activation. To uncover this aspect, we performed the following experiments. Methods and results: Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Co., Ltd. The cells were grown to 80% confluence and were treated with rivaroxaban (100nM, 500nM, 1000nM, 2000nM respectively) without FXa stimulation for 4 h, 10 h or 24 h. Cells and medium were collected and then their RNA was extracted from the cells. The qPCR of MCP-1, PAR1-4 and the DNA micro arrays (The GeneChip Human Gene 2.0 ST Array, Affymetrix) were performed. There was neither increased nor decreased gene expression significantly in either experimental time course of the qPCRs or the the DNA micro arrays. The ELISA assay of MCP-1 with medium showed non-activated MCP-1. As a next step, cells were treated with 100nM FXa and with/without rivaroxaban in same time course, and cells and medium were collected for further experiments. FXa evoked induction of mRNA levels for several pro-inflammatory cytokines including MCP-1 maximally at 4h, whereas MCP-1 was maximally evoked at 24 h in ELISA assay. Interestingly rivaroxaban inhibited both in all time course, at 4 hour inflammatory phase and at 24 hour inflammatory phase. Conclusion: Collectively, these results suggest that rivaroxaban may be safe in the inactivated coagulation state, and has the efficacy to attenuate the endothelial damage evoked by FXa and by pro-inflammatory cytokine genes.

DDR1 methylation is associated with bipolar disorder and the isoform expression and methylation of myelin genes

Epigenomics ◽  
2021 ◽  
Author(s):  
Beatriz Garcia-Ruiz ◽  
Manuel Castro de Moura ◽  
Gerard Muntané ◽  
Lourdes Martorell ◽  
Elena Bosch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bipolar Disorder ◽  
Mrna Expression ◽  
Gene Expression Analysis ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Genome Wide ◽  
Isoform Expression

Aim: To investigate DDR1 methylation in the brains of bipolar disorder (BD) patients and its association with DDR1 mRNA levels and comethylation with myelin genes. Materials & methods: Genome-wide profiling of DNA methylation (Infinium MethylationEPIC BeadChip) corrected for glial composition and DDR1 gene expression analysis in the occipital cortices of individuals with BD (n = 15) and healthy controls (n = 15) were conducted. Results: DDR1 5-methylcytosine levels were increased and directly associated with DDR1b mRNA expression in the brains of BD patients. We also observed that DDR1 was comethylated with a group of myelin genes. Conclusion: DDR1 is hypermethylated in BD brain tissue and is associated with isoform expression. Additionally, DDR1 comethylation with myelin genes supports the role of this receptor in myelination.

scholarly journals Identification of anti-norovirus genes in mouse and human cells using genome-wide CRISPR activation screening

2018 ◽  
Author(s):  
Robert C. Orchard ◽  
Meagan E. Sullender ◽  
Bria F. Dunlap ◽  
Dale R. Balce ◽  
John G. Doench ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
World Wide ◽  
Human Cells ◽  
Gene Promoters ◽  
Murine Norovirus ◽  
Individual Gene ◽  
Genome Wide ◽  
Host Genes ◽  
Crispr Activation

AbstractNoroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in either mouse or human cells. Our screens identified with high confidence 57 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity. Confirmatory studies of eight of the genes in validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules across cell types and species.Author SummaryNorovirus is one of the leading causes of foodborne illness world-wide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed we performed genome-wide CRISPR activation (CRISPRa) screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 57 genes could block murine norovirus replication in either mouse or human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data is a resource for those studying norovirus and we provide a robust approach to identify novel antiviral genes.

Expression of the iron-regulatory protein haemojuvelin in retina and its regulation during cytomegalovirus infection

2009 ◽  
Vol 419 (3) ◽  
pp. 533-543 ◽  
Author(s):  
Jaya P. Gnana-Prakasam ◽  
Ming Zhang ◽  
Pamela M. Martin ◽  
Sally S. Atherton ◽  
Sylvia B. Smith ◽  
...  
Keyword(s):  
Genetic Disorder ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Regulatory Proteins ◽  
Mouse Retina ◽  
Mrna Levels ◽  
Cmv Infection ◽  
Iron Regulatory Proteins ◽  
Rpe Cells ◽  
Gene Mrna

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE [HLA-like protein involved in iron (Fe) homoeostasis], transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Expression of the first four genes coding for these proteins in retina has been established. Here we report on the expression of HJV. Since infection of retina with CMV (cytomegalovirus) causes blindness, we also investigated the expression of HJV and other iron-regulatory proteins in retina during CMV infection. HJV (HJV gene) mRNA was expressed in RPE (retinal pigment epithelium)/eyecup and neural retina in mouse. In situ hybridization and immunohistochemistry confirmed the presence of HJV mRNA and protein in RPE, outer and inner nuclear layers, and ganglion cell layer. Immunocytochemistry with cell lines and primary cell cultures showed HJV expression in RPE and Müller cells. In RPE, the expression was restricted to apical membrane. Infection of primary cultures of mouse RPE with CMV increased HJV mRNA and protein levels. Under similar conditions, HFE (HFE gene) mRNA levels were not altered, but HFE protein was decreased. Hepcidin expression was, however, not altered. These findings were demonstrable in vivo with CMV-infected mouse retina. The CMV-induced up-regulation of HJV in RPE was independent of changes in HFE because the phenomenon was also seen in HFE-null RPE cells. CMV-infected primary RPE cells showed evidence of iron accumulation and oxidative stress, as indicated by increased levels of ferritin and hydroxynonenal. The observed changes in HJV expression and iron status during CMV infection in retina may have significance in the pathophysiology of CMV retinitis

scholarly journals Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Robert C. Orchard ◽  
Meagan E. Sullender ◽  
Bria F. Dunlap ◽  
Dale R. Balce ◽  
John G. Doench ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Entry ◽  
Human Cells ◽  
Gene Promoters ◽  
Murine Norovirus ◽  
Individual Gene ◽  
Genome Wide ◽  
Host Genes ◽  
Crispr Activation

ABSTRACT Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together, these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules. IMPORTANCE Norovirus is one of the leading causes of food-borne illness worldwide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed, we performed genome-wide CRISPR activation screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes that could block murine norovirus replication in human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data are a resource for those studying noroviruses, and we provide a robust approach to identify novel antiviral genes.

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