scholarly journals Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

2018 ◽  
Author(s):  
Claire Simonneau ◽  
Junning Yang ◽  
Xianguo Kong ◽  
Robert Kilker ◽  
Leonard Edelstein ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
High Throughput ◽  
Target Genes ◽  
Cell Monolayer ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Disruptive Effect ◽  
Genome Wide ◽  
Genes Encoding ◽  
Permeability Assay

ABSTRACTThe impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer’s intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ∼150 μm microcarriers. Each microcarrier functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of the measurement of impedance. We validate the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by a fluorescent probe from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.

scholarly journals Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Claire Simonneau ◽  
Junning Yang ◽  
Xianguo Kong ◽  
Robert Kilker ◽  
Leonard Edelstein ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
High Throughput ◽  
Target Genes ◽  
Cell Monolayer ◽  
Impedance Measurement ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Disruptive Effect ◽  
Genome Wide ◽  
Permeability Assay

Abstract The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer’s intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.

scholarly journals HSF1Base: A Comprehensive Database of HSF1 (Heat Shock Factor 1) Target Genes

2019 ◽  
Vol 20 (22) ◽  
pp. 5815 ◽  
Author(s):  
Kovács ◽  
Sigmond ◽  
Hotzi ◽  
Bohár ◽  
Fazekas ◽  
...  
Keyword(s):  
Heat Shock ◽  
High Throughput ◽  
Ribosome Biogenesis ◽  
Target Genes ◽  
Enrichment Analysis ◽  
Cell Types ◽  
Heat Shock Factor ◽  
Heat Shock Factor 1 ◽  
Genes Encoding ◽  
Shock Proteins

: HSF1 (heat shock factor 1) is an evolutionarily conserved master transcriptional regulator of the heat shock response (HSR) in eukaryotic cells. In response to high temperatures, HSF1 upregulates genes encoding molecular chaperones, also called heat shock proteins, which assist the refolding or degradation of damaged intracellular proteins. Accumulating evidence reveals however that HSF1 participates in several other physiological and pathological processes such as differentiation, immune response, and multidrug resistance, as well as in ageing, neurodegenerative demise, and cancer. To address how HSF1 controls these processes one should systematically analyze its target genes. Here we present a novel database called HSF1Base (hsf1base.org) that contains a nearly comprehensive list of HSF1 target genes identified so far. The list was obtained by manually curating publications on individual HSF1 targets and analyzing relevant high throughput transcriptomic and chromatin immunoprecipitation data derived from the literature and the Yeastract database. To support the biological relevance of HSF1 targets identified by high throughput methods, we performed an enrichment analysis of (potential) HSF1 targets across different tissues/cell types and organisms. We found that general HSF1 functions (targets are expressed in all tissues/cell types) are mostly related to cellular proteostasis. Furthermore, HSF1 targets that are conserved across various animal taxa operate mostly in cellular stress pathways (e.g., autophagy), chromatin remodeling, ribosome biogenesis, and ageing. Together, these data highlight diverse roles for HSF1, expanding far beyond the HSR.

scholarly journals Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.

1991 ◽  
Vol 114 (5) ◽  
pp. 1059-1068 ◽  
Author(s):  
S M Albelda ◽  
W A Muller ◽  
C A Buck ◽  
P J Newman
Keyword(s):  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Immunoglobulin Gene ◽  
Vascular Cell ◽  
L Cells ◽  
Cell Cell

PECAM-1 is a 130-120-kD integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. Previous studies have shown that it is a member of the immunoglobulin gene superfamily and that antibodies against the bovine form of this protein (endoCAM) can inhibit endothelial cell-cell interactions. These data suggest that PECAM-1 may function as a vascular cell adhesion molecule. The function of this molecule has been further evaluated by transfecting cells with a full-length PECAM-1 cDNA. Transfected COS-7, mouse 3T3 and L cells expressed a 130-120-kD glycoprotein on their cell surface that reacted with anti-PECAM-1 polyclonal and monoclonal antibodies. COS-7 and 3T3 cell transfectants formed cell-cell junctions that were highly enriched in PECAM-1, reminiscent of its distribution at endothelial cell-cell borders. In contrast, this protein remained diffusely distributed within the plasma membrane of PECAM-1 transfected cells that were in contact with mock transfectants. Mouse L cells stably transfected with PECAM-1 demonstrated calcium-dependent aggregation that was inhibited by anti-PECAM antibodies. These results demonstrate that PECAM-1 mediates cell-cell adhesion and support the idea that it may be involved in some of the interactive events taking place during thrombosis, wound healing, and angiogenesis.

scholarly journals QTL Mapping of Web Blotch Resistance in Peanut by High-throughput Genome-wide Sequencing

2020 ◽  
Author(s):  
Hua Liu ◽  
Ziqi Sun ◽  
Xinyou Zhang ◽  
Li Qin ◽  
Feiyan Qi ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Breeding ◽  
Phenotypic Variance ◽  
Linkage Groups ◽  
Frequency Distributions ◽  
Genome Wide ◽  
Genes Encoding ◽  
Phenotypic Variations ◽  
Disease Resistances ◽  
Disease Grade

Abstract Background: Web blotch is one of the most important foliar diseases worldwide in peanut (Arachis hypogaea L.). The identification of quantitative trait loci (QTLs) for peanut web blotch resistance represents the basis for gene mining and the application of molecular breeding technologies.Results: In this study, a peanut recombinant inbred line (RIL) population was used to map QTLs for web blotch resistance based on high-throughput genome-wide sequencing. Frequency distributions of disease grade and disease index in five environments indicated wide phenotypic variations in response to web blotch among RILs. A high-density genetic map was constructed, containing 3,634 bin markers distributed on 20 peanut linkage groups (LGs) with an average genetic distance of 0.5 cM. In total, eight QTLs were detected for peanut web blotch resistance in at least two environments, explaining from 2.8% to 15.1% of phenotypic variance. Two major QTLs qWBRA04 and qWBRA14 were detected in all five environments and were linked to 40 candidate genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) or other proteins related to disease resistances. Conclusions: The results of this study provide a basis for breeding peanut cultivars with web blotch resistance.

scholarly journals Endothelial cell junctions

1975 ◽  
Vol 66 (1) ◽  
pp. 200-204 ◽  
Author(s):  
AG Yee ◽  
JP Revel
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Rat Liver ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Intramembranous Particles ◽  
Zonula Occludens ◽  
Regenerating Rat Liver ◽  
Individual Particles

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.

scholarly journals QTL Mapping of Web Blotch Resistance in Peanut by High-throughput Genome-wide Sequencing

2020 ◽  
Author(s):  
Hua Liu ◽  
Ziqi Sun ◽  
Xinyou Zhang ◽  
Li Qin ◽  
Feiyan Qi ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Breeding ◽  
Disease Index ◽  
Phenotypic Variance ◽  
Leucine Rich Repeat ◽  
Linkage Groups ◽  
Genome Wide ◽  
Genes Encoding ◽  
Disease Resistances ◽  
Disease Grade

Abstract Background: Web blotch is one of the most important foliar diseases worldwide in peanut (Arachis hypogaea L.). The identification of quantitative trait loci (QTLs) for peanut web blotch resistance represents the basis for gene mining and the application of molecular breeding technologies.Results: In this study, a peanut recombinant inbred line (RIL) population was used to map QTLs for web blotch resistance by high-throughput genome-wide sequencing. Frequency distribution of disease grade and disease index in five environments indicated wide phenotypic variation in response to web blotch among RILs. A high-density genetic map was constructed, containing 3,634 bin markers distributed on 20 peanut linkage groups (LGs) with an average genetic distance of 0.5 cM. In total, eight QTLs were detected for peanut web blotch resistance in at least three environments, explaining from 2.8 to 15.1% of phenotypic variance. The two major QTLs qWBRA04 and qWBRA14 were detected in all five environments and were linked to 41 candidate genes encoding nucleotide-binding site leucine-rich repeat (NBS-LRR) or other proteins related to disease resistances. Conclusions: The results of this study provide a basis for breeding peanut cultivars with web blotch resistance.

scholarly journals Genome-wide approaches for identification of nuclear receptor target genes

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04018 ◽  
Author(s):  
Luz E. Tavera-Mendoza ◽  
Sylvie Mader ◽  
John H. White
Keyword(s):  
Nuclear Receptor ◽  
High Throughput ◽  
Large Scale ◽  
Target Genes ◽  
Receptor Signaling ◽  
Receptor Field ◽  
Hormone Response Element ◽  
Genome Wide ◽  
Gene Switches ◽  
On Chip

Large-scale genomics analyses have grown by leaps and bounds with the rapid advances in high throughput DNA sequencing and synthesis techniques. Nuclear receptor signaling is ideally suited to genomics studies because receptors function as ligand-regulated gene switches. This review will survey the strengths and limitations of three major classes of high throughput techniques widely used in the nuclear receptor field to characterize ligand-dependent gene regulation: expression profiling studies (microarrays, SAGE and related techniques), chromatin immunoprecipitation followed by microarray (ChIP-on-chip), and genome-wide in silico hormone response element screens. We will discuss each technique, and how each has contributed to our understanding of nuclear receptor signaling.

scholarly journals Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling

2009 ◽  
Vol 296 (6) ◽  
pp. R1726-R1734 ◽  
Author(s):  
Mohammad N. Uddin ◽  
Darijana Horvat ◽  
Ed W. Childs ◽  
Jules B. Puschett
Keyword(s):  
Endothelial Cell ◽  
Sodium Pump ◽  
Cell Monolayer ◽  
Endothelial Permeability ◽  
Annexin V ◽  
Cell Junctions ◽  
P38 Inhibitor ◽  
Caspase Inhibition ◽  
Monolayer Permeability ◽  
Induced Apoptosis

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na+/K+-ATPase. Increased plasma levels of MBG have been reported in patients with preeclampsia. MBG increases microvascular barrier permeability in an animal model of preeclampsia. However, the mechanism by which MBG impairs endothelial permeability is unknown. We utilized rat lung microvascular endothelial cells (RLMEC) to examine alterations in MBG-induced monolayer permeability and the effect of MBG on the phosphorylation status of ERK1/2, Jnk, and p38. Apoptosis was evaluated by examining alterations in caspases 3/7, 8, and 9 and annexin-V staining. We also examined the effect of MBG on the endothelial adherens junctions of the RLMEC monolayer. MBG inhibited the proliferation, and increased the monolayer permeability, of RLMEC. These actions of MBG were attenuated by ERK, p38, and pan caspase inhibition. MBG significantly decreased the phosphorylation of ERK1/2 and activated the phosphorylation of Jnk and p38. MBG also significantly increased the expression of caspases 3/7, 8, and 9, indicating the activation of apoptosis. MBG-induced apoptosis signaling was not observed in cells pretreated with a p38 inhibitor. MBG treatment induced the disruption of endothelial cell junctions. This effect was prevented by a pan caspase inhibitor. In conclusion, 1) MBG induced an impairment of RLMEC proliferation; 2) the bufadienolide also caused endothelial hyperpermeability; and 3) these effects of MBG were mediated by the downregulation of ERK1/2, the upregulation of Jnk and p38, by the activation of apoptosis, and by the disruption of endothelial cell junctions.

Essential Role for Activated Leukocyte Cell Adhesion Molecule in Transendothelial Migration.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3691-3691
Author(s):  
Solomon F. Ofori-Acquah
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Essential Role ◽  
Adherens Junction ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Mononuclear Leukocytes

Abstract Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin cell adhesion super family, which has been implicated in diverse physiological and pathophysiological events involving cell migration. Hitherto, ALCAM’s role in inflammation has not been determined. In this study, we show ALCAM is involved in controlling migration of mononuclear leukocytes across the pulmonary endothelium. We demonstrated that ALCAM is localized at intercellular junctions in pulmonary microvascular endothelial cells in vitro and in vivo. ALCAM co-localized with multiple adherens junction molecules including cadherins, catenins and Dlg, as determined by confocal microscopy, and these observations were confirmed by co-immunoprecipitation and co-distribution assays. Treatment of endothelial cultures with EGTA and cytochalasin D translocated ALCAM from intercellular junctions to the cytosol indicating a requirement for homotypic cadherin adhesion and an intact endothelial cytoskeleton for maintaining ALCAM at endothelial cell junctions. Collectively, these data supports the conclusion that ALCAM contributes to the adherens junction complex in endothelial cells. To determine ALCAM’s role in leukocyte-endothelial cell interactions, adult Sprague Dawley rats were intratracheally instilled with macrophage inflammatory protein-1, and this treatment caused acute expression of ALCAM exclusively in newly recruited mononuclear but not polymorphonuclear leukocytes in the alveolar airway. Given that no ALCAM reactivity was observed in peripheral blood leukocytes, we concluded ALCAM is activated as part of the phenotypic switch by mononuclear leukocytes transitioning from circulation to interstitial tissue compartments. To determine the physiological relevance of this finding we examined whether ALCAM was required for transendothelial migration using monocyte chemoattractant protein 1 (MCP-1). MCP-1 dose- and time-dependently increased the number of transmigrated THP-1 monocytes across pulmonary microvascular endothelial monolayers. Recombinant soluble ALCAM dose-dependently reduced the number of transmigrated THP-1 monocytes, whereas in control experiments recombinant soluble vascular endothelial cadherin had no effect on transmigration. This study shows for the first time that ALCAM is located at endothelial cell junctions where it is intimately involved in controlling the number of monocytes that pass through endothelial barriers. ALCAM may therefore play an essential role in the response to inflammation by enhancing recruitment of mononuclear leukocytes by inflamed tissues.

scholarly journals Delineation of the Salmonella enterica Serovar Typhimurium HilA Regulon through Genome-Wide Location and Transcript Analysis

2007 ◽  
Vol 189 (13) ◽  
pp. 4587-4596 ◽  
Author(s):  
Inge M. V. Thijs ◽  
Sigrid C. J. De Keersmaecker ◽  
Abeer Fadda ◽  
Kristof Engelen ◽  
Hui Zhao ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Target Genes ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Effector Proteins ◽  
Transcript Analysis ◽  
Genome Wide ◽  
Serovar Typhimurium ◽  
Genes Encoding

ABSTRACT The Salmonella enterica serovar Typhimurium HilA protein is the key regulator for the invasion of epithelial cells. By a combination of genome-wide location and transcript analysis, the HilA-dependent regulon has been delineated. Under invasion-inducing conditions, HilA binds to most of the known target genes and a number of new target genes. The sopB, sopE, and sopA genes, encoding effector proteins secreted by the type III secretion system on Salmonella pathogenicity island 1 (SPI-1), were identified as being both bound by HilA and differentially regulated in an HilA mutant. This suggests a cooperative role for HilA and InvF in the regulation of SPI-1-secreted effectors. Also, siiA, the first gene of SPI-4, is both bound by HilA and differentially regulated in an HilA mutant, thus linking this pathogenicity island to the invasion key regulator. Finally, the interactions of HilA with the SPI-2 secretion system gene ssaH and the flagellar gene flhD imply a repressor function for HilA under invasion-inducing conditions.

Sign in / Sign up

Export Citation Format

Share Document