scholarly journals CFTR mutations altering CFTR fragmentation

2012 ◽  
Vol 449 (1) ◽  
pp. 295-305 ◽  
Author(s):  
Kendra Tosoni ◽  
Michelle Stobbart ◽  
Diane M. Cassidy ◽  
Andrea Venerando ◽  
Mario A. Pagano ◽  
...  
Keyword(s):  
Bronchial Epithelial Cell ◽  
Epithelial Cell Line ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Ladder Pattern ◽  
Cftr Mutations ◽  
Cf Transmembrane Conductance Regulator ◽  
In Vitro Interaction ◽  
Kinase Ck2

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 867
Author(s):  
Olga Povolyaeva ◽  
Yaroslava Chalenko ◽  
Egor Kalinin ◽  
Olga Kolbasova ◽  
Elena Pivova ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Domestic Animals ◽  
Epithelial Cell Line ◽  
Intracellular Pathogen ◽  
Wild Type ◽  
Listeria Monocytogenes Infection ◽  
Natural Hosts

L. monocytogenes is a widespread facultative intracellular pathogen. The range of natural hosts that supporting L. monocytogenes persistence in the environment has not been fully established yet. In this study, we were interested in the potential of L. monocytogenes to infect cells of bats, which are being increasingly recognized as a reservoir for microorganisms that are pathogenic to humans and domestic animals. A stable epithelial cell line was developed from the kidneys of Pipistrellus nathusii, a small bat widely distributed across Europe. The wild-type L. monocytogenes strain EGDe infected this cell line with an invasion efficiency of 0.0078 ± 0.0009%. Once it entered bat cells, L. monocytogenes doubled within about 70 min. When L. monocytogenes lacked either of the major invasion factors, InlA and InlB, invasion efficiency decreased by a factor of 10 and 25 respectively (p < 0.000001). The obtained results suggest that bat epithelial cells are susceptible to L. monocytogenes infection and that L. monocytogenes invasion of bat cells depends on the major invasion factors InlA and InlB. These results constitute the first report on in vitro studies of L. monocytogenes infection in bats.

scholarly journals Expression/Activation of PAR-1 in Airway Epithelial Cells of COPD Patients: Ex Vivo/In Vitro Study

2021 ◽  
Vol 22 (19) ◽  
pp. 10703
Author(s):  
Angela Marina Montalbano ◽  
Giuseppina Chiappara ◽  
Giusy Daniela Albano ◽  
Maria Ferraro ◽  
Caterina Di Sano ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
In Vitro Study ◽  
Bronchial Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Copd Patients

The role of PAR-1 expression and activation was described in epithelial cells from the central and distal airways of COPD patients using an ex vivo/in vitro model. PAR-1 immunoreactivity was studied in epithelial cells from surgical specimens of the central and distal airways of COPD patients and healthy control (HC). Furthermore, PAR-1 expression and activation were measured in both the human bronchial epithelial cell line (16HBE) and normal human bronchial epithelial cells (NHBEs) exposed to cigarette smoke extract (CSE) (10%) or thrombin. Finally, cell proliferation, apoptosis, and IL-8 release were detected in stimulated NHBEs. We identified higher levels of PAR-1 expression/activation in epithelial cells from the central airways of COPD patients than in HC. Active PAR-1 increased in epithelial cells from central and distal airways of COPD, with higher levels in COPD smokers (correlated with pack-years) than in COPD ex-smokers. 16HBE and NHBEs exposed to CSE or thrombin showed increased levels of active PAR-1 (localized in the cytoplasm) than baseline conditions, while NHBEs treated with thrombin or CSE showed increased levels of IL-8 proteins, with an additional effect when used in combination. Smoking habits generate the upregulation of PAR-1 expression/activation in airway epithelial cells, and promoting IL-8 release might affect the recruitment of infiltrating cells in the airways of COPD patients.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 153
Author(s):  
Z. Fan ◽  
Y. Liu ◽  
I. V. Perisse ◽  
K. L. White ◽  
I. A. Polejaeva
Keyword(s):  
Cftr Gene ◽  
Transmembrane Conductance Regulator ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Significant Difference ◽  
Cftr Mutations ◽  
Developmental Capacity ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P&gt;0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

scholarly journals Correction of a CFTR/G542X Mutation Using CRISPR/Cas9 Editing in Ovine-bovine Interspecies Embryos

2021 ◽  
Author(s):  
Zhiqiang Fan ◽  
Ying Liu ◽  
Iuri Perisse ◽  
Ann Harris ◽  
Kenneth White ◽  
...  
Keyword(s):  
Transmembrane Conductance Regulator ◽  
Gene Correction ◽  
Human Genetic Disease ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Stage Embryo ◽  
Developmental Capacity ◽  
Embryo Stage ◽  
Cf Transmembrane Conductance Regulator

Abstract Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Correction of CFTR mutations at embryo stage could be a permanent solution to cure this disease. To assess the efficiency of CFTR/G542X mutation correction in vitro by CRISPR/Cas9, we utilized embryos generated by ovine-bovine interspecies somatic cell nuclear transfer (iSCNT) due to a limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos. These embryos were able to develop to 16-cell stage, allowing for individual embryo genotyping. Then, we optimized the concentrations of Cas9:gRNA ribonucleoprotein (RNP) for 1-cell stage embryo injection. Genotyping results showed that we achieved high efficiencies (88.9–100%) of indel mutations at the target locus after injection of different concentrations of RNP. When an RNP (0.09 µg/µl:2.3 µM) was co-injected with a ssODN (18 µM), the G542X mutation was corrected via the homology-directed repair in 11.1% (1/9) of iSCNT embryos. Taken together, we developed an effective strategy to correct the CFTR/G542X mutation in ovine-bovine iSCNT embryos by CRISRP/Cas9. Our strategy overcomes the limitation of oocyte source and provides the opportunity of mimicking the editing of any other genes in one-cell embryos of different species.

scholarly journals Role of Aspergillus fumigatus DvrA in Host Cell Interactions and Virulence

2010 ◽  
Vol 9 (10) ◽  
pp. 1432-1440 ◽  
Author(s):  
Daniele E. Ejzykowicz ◽  
Norma V. Solis ◽  
Fabrice N. Gravelat ◽  
Josee Chabot ◽  
Xuexian Li ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Type Strain ◽  
Wild Type Strain ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Wild Type ◽  
Content Type ◽  
Pulmonary Epithelial Cell

ABSTRACT The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.

scholarly journals COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code

2004 ◽  
Vol 167 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Xiaodong Wang ◽  
Jeanne Matteson ◽  
Yu An ◽  
Bryan Moyer ◽  
Jin-San Yoo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Hereditary Disease ◽  
Mutant Form ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Primary Defect ◽  
Cf Transmembrane Conductance Regulator ◽  
Er Export ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) ΔF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in ΔF508-expressing patients.

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