22 results on '"Git Chung"'
Search Results
2. The chlorophenoxy herbicide MCPA: a mechanistic basis for the observed differences in toxicological profile in humans and rats versus dogs
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Alex Gledhill, Rachael Bowen, Michael Bartels, Andrew Bond, Git Chung, Colin Brown, Keith Pye, and Tarang Vora
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Pharmacology ,Herbicides ,Health, Toxicology and Mutagenesis ,General Medicine ,2-Methyl-4-chlorophenoxyacetic Acid ,Toxicology ,Biochemistry ,Rats ,Feces ,Kinetics ,Dogs ,Species Specificity ,Animals ,Humans - Abstract
Metabolism data for MCPA in rat, dog and human shows a single oral dose is quantitatively and rapidly absorbed with evidence of non-linear kinetics at100 mg/kg bw. The extent of metabolism is low and consistent between rat and human, with substantially higher metabolic conversion in dog. Parent accounts for 50%-67% dose in rat, ∼40% in human and 2%-27% in dog. No dog specific metabolite is apparent.In rat and human, MCPA and metabolites are rapidly eliminated in urine (65%-70% within 24 h) but in dog, excretion is
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- 2022
3. Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (
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Hany S, Zinad, Chanachai, Sae-Lee, Maria Ascensión, Ariza-Mateos, Grace, Adamson, Mushtaq Mufleh, Khazeem, Amber, Knox, Git, Chung, Jelena, Mann, and Andreas, Werner
- Abstract
Natural antisense transcripts (NATs) constitute a significant group of regulatory, long noncoding RNAs. They are prominently expressed in testis but are also detectable in other organs. NATs are transcribed at low levels and co-expressed with related protein coding sense transcripts. Nowadays NATs are generally considered as regulatory, long noncoding RNAs without closer focus on the inevitable interference between sense and antisense expression. This work describes a cellular system where sense and antisense transcription of a specific locus (
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- 2021
4. SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells
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Git Chung, Edward I Patterson, Maria Georgiou, Grant L. Hughes, Majlinda Lako, Shaun H. Pennington, Ivo Djidrovski, Lyle Armstrong, Martine J. Smit, Jelle van den Bor, Aitor Casas-Sanchez, Giancarlo A. Biagini, Marina Moya-Molina, Medicinal chemistry, and AIMMS
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0301 basic medicine ,Cell type ,induced pluripotent stem cells ,Biology ,Virus Replication ,Models, Biological ,Cell Line ,Embryonic Stem Cells/Induced Pluripotent Stem Cells ,lung ,03 medical and health sciences ,0302 clinical medicine ,SDG 3 - Good Health and Well-being ,In vivo ,medicine ,Humans ,Secretion ,Respiratory system ,Induced pluripotent stem cell ,Lung ,SARS-CoV-2 ,COVID-19 ,Epithelial Cells ,Cell Biology ,respiratory system ,interleukins (ILs) ,Mucus ,cytokines ,respiratory tract diseases ,3. Good health ,Cell biology ,interleukins ,030104 developmental biology ,medicine.anatomical_structure ,Molecular Medicine ,Respiratory epithelium ,Inflammation Mediators ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal‐like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air‐liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs., We have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways that are capable of supporting SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways.
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- 2021
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5. Freshly isolated primary human proximal tubule cells as an in vitro model for the detection of renal tubular toxicity
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Tomoya Yukawa, Akio Imanishi, Piyush Bajaj, Matthew Wagoner, Colin D. Brown, Yuichi Takai, Keith Pye, and Git Chung
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0301 basic medicine ,Organic anion transporter 1 ,Endpoint Determination ,Primary Cell Culture ,Gene Expression ,Lipocalin ,Pharmacology ,Toxicology ,Nephrotoxicity ,Kidney Tubules, Proximal ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Kidney ,Clusterin ,biology ,Chemistry ,Organic Cation Transporter 2 ,Reproducibility of Results ,Transporter ,Cubilin ,Fanconi Syndrome ,030104 developmental biology ,medicine.anatomical_structure ,Toxicity ,biology.protein ,030217 neurology & neurosurgery ,Biomarkers ,Octamer Transcription Factor-1 - Abstract
Drug induced kidney injury (DIKI) is a common reason for compound attrition in drug development pipelines with proximal tubule epithelial cells (PTECs) most commonly associated with DIKI. Here, we investigated freshly isolated human (hPTECs) as an in vitro model for assessing renal tubular toxicity. The freshly isolated hPTECs were first characterized to confirm gene expression of important renal transporters involved in drug handling which was further corroborated by confirming the functional activity of organic cation transporter 2 and organic anion transporter 1 by using transporter specific inhibitors. Additionally, functionality of megalin/cubilin endocytic receptors was also confirmed. A training set of 36 compounds was used to test the ability of the model to classify them using six different endpoints which included three biomarkers (Kidney Injury Molecule-1, Neutrophil gelatinase-associated lipocalin, and Clusterin) and three non-specific injury endpoints (ATP depletion, LDH leakage, and barrier permeability via transepithelial electrical resistance) in a dose-dependent manner across two independent kidney donors. In general, biomarkers showed higher predictivity than non-specific endpoints, with Clusterin showing the highest predictivity (Sensitivity/Specificity - 65.0/93.8 %). By using the thresholds generated from the training set, nine candidate internal Takeda compounds were screened where PTEC toxicity was identified as one of the findings in preclinical animal studies. The model correctly classified four of six true positives and two of three true negatives, showing validation of the in vitro model for detection of tubular toxicants. This work thus shows the potential application of freshly isolated primary hPTECs using translational biomarkers in assessment of tubular toxicity within the drug discovery pipeline.
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- 2020
6. Review of the pharmacokinetics and metabolism of triclopyr herbicide in mammals: Impact on safety assessments
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Colin D. Brown, Git Chung, Melissa Chan, Michael J. Bartels, Marco Corvaro, Claire Terry, and Sean C. Gehen
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Organic anion transporter 1 ,Triclopyr ,Absorption (skin) ,010501 environmental sciences ,Pharmacology ,Toxicology ,030226 pharmacology & pharmacy ,01 natural sciences ,Risk Assessment ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Pharmacokinetics ,Toxicokinetics ,Animals ,Humans ,0105 earth and related environmental sciences ,biology ,Reabsorption ,Chemistry ,Herbicides ,General Medicine ,Glycolates ,Free fraction ,Toxicity ,biology.protein - Abstract
A review of pharmacokinetic and metabolism studies show that triclopyr is well absorbed from the oral route in numerous species (≥80%), primarily as parent compound. Absorption is quite rapid in rats, dogs and human volunteers. Plasma or blood clearance is also rapid (t1/2 3–9 h), except for dog (12–96 h). Systemic exposure is not dose-proportional: in the rat above 20 mg/kg (dietary) or between 3 and 60 mg/kg (gavage), or in dogs above 5 mg/kg, with systemic exposure in human more comparable to rat than dog. Triclopyr is highly bound to protein in rat, dog and human plasma (≥97% at or below 7 μg/mL), indicating that species differences in systemic exposure are not due to differences in the free fraction of this test material in plasma. An in vitro flux study in renal proximal tubule cells showed that net renal transport of triclopyr is in the direction of secretion in rat and human donors, while reabsorption predominated in the dog, possibly via organic anion transporters such as OAT1/3. These results fit well into the framework of utilizing metabolism and toxicokinetics across species and exposure levels to allow for toxicity testing in the most relevant species as well as at proper dose levels.
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- 2020
7. c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis
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Johannes L Zakrzewski, Ben S. Barksby, Jeremy French, Luc Schoonjans, Amy L Collins, Jelena Mann, Matthias Trost, Stuart Robinson, Ulf Klein, Morten A. Karsdal, Hannah L Paish, Amber Knox, Peter Carmeliet, Lee A. Borthwick, Andrew D Blanchard, Git Chung, Rainie Cameron, Neil S. Sheerin, Laure-Anne Teuwen, Ingmar Mederacke, Lucy M Gee, Colin D.A. Brown, Carmel B. Nanthakumar, Thomas G. Bird, Jack Leslie, Sandra Murphy, Robert F. Schwabe, Fiona Oakley, Marina García Macia, Xin Xu, Andrew J. Fisher, Derek A. Mann, Derek Manas, Rachel A. Burgoyne, William J Reilly, Steven A. White, Charlotte Bragg, Saimir Luli, Gourab Sen, Marco Y W Zaki, Colin Nixon, and Julie C. Worrell
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Liver Cirrhosis ,Cell signaling ,Phosphofructokinase-2 ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Liver fibrosis ,Mitosis ,Connective tissue ,Epithelium ,Article ,Mice ,Paracrine signalling ,Fibrosis ,Physiology (medical) ,Paracrine Communication ,Internal Medicine ,medicine ,Animals ,Macrophage ,Monocytes and macrophages ,Mice, Knockout ,Chemistry ,Macrophages ,Growth factor ,Mesenchymal stem cell ,Cell Polarity ,Cell Biology ,medicine.disease ,Proto-Oncogene Proteins c-rel ,Liver Regeneration ,Cell biology ,Mice, Inbred C57BL ,Hydroxyproline ,medicine.anatomical_structure ,Metabolism ,Gene Targeting ,Hepatocytes ,REL ,Cell signalling - Abstract
Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-β1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease. ispartof: NATURE METABOLISM vol:2 issue:11 ispartof: location:Germany status: published
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- 2020
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8. Derivation of a System-IndependentKifor P-glycoprotein Mediated Digoxin Transport from System-Dependent IC50Data
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Git Chung, Colin D.A. Brown, Joe Bentz, Akshata Yalvigi, Caroline Lee, Michael O'Connor, Adam Lynn, Harma Ellens, and Aqsaa Chaudhry
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0301 basic medicine ,Pharmacology ,biology ,Digoxin ,Chemistry ,OATP4C1 ,Pharmaceutical Science ,ATP-binding cassette transporter ,Transporter ,030226 pharmacology & pharmacy ,Ouabain ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Caco-2 ,Cell culture ,biology.protein ,medicine ,Biophysics ,P-glycoprotein ,medicine.drug - Abstract
It has been previously demonstrated that IC50 values for inhibition of digoxin transport across confluent polarized cell monolayers are system-dependent. Digoxin IC50 data from five laboratories participating in the P-glycoprotein (P-gp) IC50 Initiative, using Caco-2, MDCKII-hMDR1 or LLC-PK1-hMDR1 cells, were fitted by the structural mass action kinetic model for P-gp-mediated transport across confluent cell monolayers. We determined their efflux-active P-gp concentration [T(0)], inhibitor elementary dissociation rate constant from P-gp (krQ), digoxin basolateral uptake clearance (kB), and inhibitor binding affinity to the digoxin basolateral uptake transporter (KQB). We also fitted the IC50 data for inhibition of digoxin transport through monolayers of primary human proximal tubule cells (HPTCs). All cell systems kinetically required a basolateral uptake transporter for digoxin, which also bound to all inhibitors. The inhibitor krQ was cell system-independent, thereby allowing calculation of a system-independent Ki. The variability in efflux-active P-gp concentrations and basolateral uptake clearances in the five laboratories was about an order of magnitude. These laboratory-to-laboratory variabilities can explain more than 60% of the IC50 variability found in the principal component analysis plot in a previous study, supporting the hypothesis that the observed IC50 variability is primarily due to differences in expression levels of efflux-active P-gp and the basolateral digoxin uptake transporter. HPTCs had 10- to 100-fold lower efflux-active P-gp concentrations than the overexpressing cell lines, whereas their digoxin basolateral uptake clearances were similar. HPTC basolateral uptake of digoxin was inhibited 50% by 10 μM ouabain, suggesting involvement of OATP4C1.
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- 2018
9. Human iPSC-derived retinal organoid model for in vitro toxicity screening
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F. Pognan, L. Armstrong, G. Hilgen, M. Lako, Git Chung, J. Ajeian, Philip Hewitt, V. Chichagova, J. Collin, R. Queen, B. Dorgau, M. Georgiou, M Carter, M Schmitt, C. de Santis, Stefan Kustermann, Marina Moya-Molina, and E. Sernagor
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chemistry.chemical_compound ,chemistry ,Toxicity ,Organoid ,Cancer research ,Retinal ,General Medicine ,Biology ,Toxicology ,In vitro - Published
- 2021
10. Author Correction: c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis
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Luc Schoonjans, Git Chung, Rainie Cameron, Hannah L Paish, Rachel A. Burgoyne, Colin Nixon, Julie C. Worrell, Steven A. White, Matthias Trost, Derek Manas, Thomas G. Bird, Xin Xu, Stuart Robinson, Andrew J. Fisher, Ingmar Mederacke, Charlotte Bragg, Saimir Luli, Lucy M Gee, Ben S. Barksby, Colin D.A. Brown, Jack Leslie, Jeremy French, Neil S. Sheerin, Amy L Collins, Morten A. Karsdal, Andrew D Blanchard, Marco Y W Zaki, Gourab Sen, Robert F. Schwabe, Fiona Oakley, Peter Carmeliet, Amber Knox, Marina García Macia, Carmel B. Nanthakumar, Ulf Klein, Laure-Anne Teuwen, Johannes L Zakrzewski, Sandra Murphy, Lee A. Borthwick, Jelena Mann, Derek A. Mann, and William J Reilly
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Energy dependent ,Cell signaling ,business.industry ,Endocrinology, Diabetes and Metabolism ,Liver fibrosis ,Human kidney ,Cell Biology ,medicine.disease ,Fibrosis ,Physiology (medical) ,Internal Medicine ,Cancer research ,Medicine ,Macrophage ,business ,REL ,Reprogramming - Abstract
Correction to: Nature Metabolism https://doi.org/10.1038/s42255-020-00306-2, published online 9 November 2020. In the version of this article initially published, in the ×40 diseased human kidney images in Supplementary Fig. 1, the FSGS image duplicated the DN image. The error has been corrected in the HTML version of the article.
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- 2020
11. High molecular weight hyaluronic acid: a two-pronged protectant against infection of the urogenital tract?
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Judith Hall, Robert Pickard, Catherine Mowbray, Syema Shams, Anna Stanton, Git Chung, Andrejus Suchenko, Phillip D. Aldridge, and Ased Ali
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0301 basic medicine ,medicine.drug_class ,Immunology ,Antibiotics ,030232 urology & nephrology ,Biology ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Hyaluronic acid ,medicine ,Immunology and Allergy ,Escherichia coli ,General Nursing ,Innate immune system ,CD44 ,Symptomatic relief ,Epithelium ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,biology.protein ,Flagellin - Abstract
Objectives Recurrent urinary tract infections are associated with uropathogenic Escherichia coli (UPEC) ascending and infecting the urinary tract. Antibiotics provide only symptomatic relief, not prevent recurrence. Clinical evidence suggests that intravesical glycosaminoglycan therapy, such as hyaluronic acid (HA), helps reduce UTI recurrence. This has been investigated here using in vitro systems modelling the urogenital tract tissues. Methods RT4 bladder cells were preconditioned with high molecular weight HA (> 1500 kDa) at 2 mg mL-1 and challenged with UPEC to analyse barrier protection and bacterial adherence. Untreated and HA-preconditioned VK2 E6/E7 vaginal cells were challenged with E. coli flagellin (50 ng mL-1) to mimic bacterial challenge, and media analysed for lipocalin-2, human β-defensin 2 and interleukin-8 by ELISA. Experiments were repeated after siRNA knockdown of Toll-like receptors 2, 4 and 5, and CD44 to investigate signalling. Results Microscopic analyses showed reduced bacterial adherence and urothelial disruption with HA, suggesting that HA functions as a barrier protecting the epithelium from bacterial infection. Cells treated with HA and flagellin simultaneously produced more of the host antimicrobial peptide LCN2 and pro-inflammatory IL-8 (P
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- 2018
12. Human iPSC-Derived RPE and Retinal Organoids Reveal Impaired Alternative Splicing of Genes Involved in Pre-mRNA Splicing in PRPF31 Autosomal Dominant Retinitis Pigmentosa Type 11
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Sushma Nagaraja Grellscheid, Colin A. Johnson, Yuchun Ding, Lyle Armstrong, Alastair Droop, Sudeep Mehrotr, Git Chung, Revital Bronstei, Chris F. Inglehearn, Katarzyna Szymanska, Jumana Y. Al-Aama, Kathryn White, Valeria Chichagova, David H. Steel, Robin R Ali, Dean Hallam, Martin McKibbin, Lili Zhu, Adriana Buskin, Eric A. Pierce, Majlinda Lako, Yaobo Xu, Stefan Przyborski, Reinhard Lührmann, Michael H. Farkas, Sameer E. Al-Harthi, Carla Mellough, Sina Mozaffari-Jovin, Gabrielle Wheway, David J. Elliott, David Dolan, Gerrit Hilgen, Basudha Basu, Susan Lindsay, Natalio Krasnogor, Katarzyna Bialas, Evelyne Sernagor, and Joseph Colli
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PRPF31 ,Retinal pigment epithelium ,Cilium ,Alternative splicing ,Retinal ,Biology ,Phenotype ,Cell biology ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,RNA splicing ,medicine ,sense organs ,Induced pluripotent stem cell - Abstract
Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.
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- 2018
13. Characterization of cisplatin toxicity in aproximate human proximal tubule cell monolayers
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Git Chung, Colin D. Brown, Mike Nicholds, and Lyle Armstrong
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Pharmacology ,Cisplatin ,medicine.anatomical_structure ,Chemistry ,Cell ,Monolayer ,Toxicity ,medicine ,Pharmaceutical Science ,Pharmacology (medical) ,Proximal tubule ,Molecular biology ,medicine.drug - Published
- 2019
14. Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa
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Adriana Buskin, Lili Zhu, Valeria Chichagova, Basudha Basu, Sina Mozaffari-Jovin, David Dolan, Alastair Droop, Joseph Collin, Revital Bronstein, Sudeep Mehrotra, Michael Farkas, Gerrit Hilgen, Kathryn White, Dean Hallam, Katarzyna Bialas, Git Chung, Carla Mellough, Yuchun Ding, Natalio Krasnogor, Stefan Przyborski, Jumana Al-Aama, Sameer Alharthi, Yaobo Xu, Gabrielle Wheway, Katarzyna Szymanska, Martin McKibbin, Chris F Inglehearn, David J Elliott, Susan Lindsay, Robin R Ali, David H Steel, Lyle Armstrong, Evelyne Sernagor, Eric Pierce, Reinhard Lüehrmann, Sushma-Nagaraja Grellscheid, Colin A Johnson, and Majlinda Lako
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0303 health sciences ,PRPF31 ,Retinal pigment epithelium ,Cilium ,030305 genetics & heredity ,Alternative splicing ,Retinal ,Biology ,Phenotype ,eye diseases ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,RNA splicing ,medicine ,sense organs ,Induced pluripotent stem cell ,030304 developmental biology - Abstract
SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes
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- 2017
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15. Derivation of a System-Independent
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Aqsaa, Chaudhry, Git, Chung, Adam, Lynn, Akshata, Yalvigi, Colin, Brown, Harma, Ellens, Michael, O'Connor, Caroline, Lee, and Joe, Bentz
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Digoxin ,Inhibitory Concentration 50 ,Kinetics ,Swine ,Cell Line, Tumor ,Animals ,Humans ,LLC-PK1 Cells ,Biological Transport ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Caco-2 Cells ,Cell Line - Abstract
It has been previously demonstrated that IC
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- 2017
16. Utility of B-13 Progenitor-Derived Hepatocytes in Hepatotoxicity and Genotoxicity Studies
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Git Chung, Steven A. White, Simon Cockell, Pasquale Mosesso, Colin D.A. Brown, Philip M. E. Probert, Loranne Agius, Matthew C. Wright, and Fiona Oakley
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Male ,cytochrome P450 ,Cellular differentiation ,Cell Culture Techniques ,Biology ,Toxicology ,liver ,Animal Testing Alternatives ,Transfection ,Dexamethasone ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Cytochrome P-450 Enzyme System ,Toxicity Tests ,medicine ,transporters ,Animals ,Induced pluripotent stem cell ,Embryonic Stem Cells ,030304 developmental biology ,Cell Proliferation ,0303 health sciences ,Sex Characteristics ,Cell growth ,Mutagenicity Tests ,Cell Differentiation ,Molecular biology ,Embryonic stem cell ,xenobiotic metabolism ,Rats ,stem cell ,medicine.anatomical_structure ,Cell culture ,030220 oncology & carcinogenesis ,Hepatocyte ,Enzyme Induction ,Hepatocytes ,Stem cell ,Research Article ,DNA Damage ,Mutagens - Abstract
Hepatocytes are the primary defining cell of the liver, performing the vast majority of its functions (Wallace et al., 2010a). Isolation and/or culture of hepatocytes are therefore common experimental techniques employed in the study of liver functions (Wallace et al., 2010a). Several factors limit hepatocyte utility. Hepatocytes do not proliferate in vitro and therefore cannot be expanded in vitro. Furthermore, culture results in dedifferentiation and loss of function. Complex culture modifications can ameliorate this loss but experimentally introduce an array of often uncharacterized factors, which can complicate interpretation (Wallace et al., 2010a). In many cases, human hepatocytes would be the ideal species to use for most experiments related to toxicity. However, human liver is in short supply and is often of poor quality. In the absence of sufficient human hepatocytes from donor livers, the main alternative for generating human hepatocytes is through differentiation of human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) (Lavon et al., 2004; Rashid et al., 2010; Takayama et al., 2012; Zhang et al., 2013). Despite significant progress over the last 12 years, it has not been possible to generate hepatocytes with function quantitatively similar to adult human hepatocytes (Takayama et al., 2012). Stem cell–derived hepatocytes remain in a fetal state and cannot progress further unless transplanted in vivo (Lavon et al., 2004; Rashid et al., 2010; Takayama et al., 2012; Yu et al., 2012; Zhang et al., 2013). Because normal hepatocytes dedifferentiate into a fetal state in vitro (even when present within culture tissue slices) (Wallace et al., 2010a; Wright and Paine, 1992), it may be not be surprising that this barrier exists in stem cell–derived hepatocyte culture. In addition, a major hurdle to the use of ESC/iPSC-derived hepatocyte is their high cost of generation. As part of an European Commission–funded project, the cost of hepatocytes derived from iPSCs was calculated, taking into consideration the most recent research that compared drug metabolism activity in iPSC-derived hepatocytes with human hepatocytes (Takayama et al., 2012). In order to obtain the hepatocytes, the iPSCs required a 4-stage differentiation protocol with a variety of recombinant growth factors. In addition, the cells were infected with adenovirus directed to overexpress 2 transcription factors. Considering growth factors alone (without taking into account the cost of culture media, virus production, culture ware, and the cost of failures), it was calculated that the cost of generating hepatocytes is approximately 5 million times greater than using glucocorticoid and a human cell line with B-13 properties (d-LIVER consortium, 2012). With current technologies, it is unlikely that ESC- or iPSC-derived hepatocytes will be a practical solution in routine in vitro toxicity testing. The B-13 cell could offer a potential route to delivering a cost-effective, simple solution to the production of functional hepatocytes in vitro. This rat pancreatic acinar-like cell line is readily expandable in simple culture medium and in response to one simple glucocorticoid hormone differentiates into nonproliferative hepatocyte-like (B-13/H) cells (Shen et al., 2000; Wallace et al., 2010b). B-13 cells model a pathophysiological response of rodent and human acinar tissue to differentiate into hepatocytes both in vitro and in vivo on exposure to high levels of glucocorticoid (Fairhall et al., 2013b; Wallace et al., 2009, 2010c). The cells retain a degree of biological stability in that they have maintained a normal karyotype in terms of the number of chromosomes per cell (although with some cytogenetic abnormalities); do not grow in soft agar (in contrast to tumorigenic cells); and are able to selectively engraft into the pancreas and liver (Fairhall et al., 2013a). Within the liver, B-13 cells differentiate into hepatocytes (Fairhall et al., 2013a). The intrinsic value of this cell line is that it offers an unlimited and reproducible supply of hepatocytes in vitro, without the requirement for tissue donors. Given both the simplicity and costs of a B-13 approach to hepatocyte generation, it is likely that a human B-13 equivalent would have utility in both experimental studies (eg, toxicity screening) and clinical applications (eg, extracorporeal liver support). In this paper, we demonstrate that B-13/H cells recapitulate the sexually dimorphic expression of cytochrome P450 (CYP)2C11; retain CYP1-3 family induction to classic inducers; and express variable levels of functional transporter expression. Stable transfection of the human CYP1A2 gene resulted in a transgenic cell line with active human protein that metabolized a probe substrate and the progenotoxin 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to a genotoxic product.
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- 2013
17. Chapter 4. Drug Transporters in the Kidney
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Git Chung, Colin D.A. Brown, Sarah Billington, and Sarah Jenkinson
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Drug ,Kidney ,media_common.quotation_subject ,Transporter ,Biology ,In vitro ,Nephrotoxicity ,Solute carrier family ,Renal Elimination ,medicine.anatomical_structure ,Biochemistry ,medicine ,Function (biology) ,media_common - Abstract
With a high expression of both uptake and efflux transporters, together with metabolic enzymes, the proximal tubule in the kidney plays a major role in determining the absorption, distribution, metabolism and elimination of a wide range of molecules. Since most members of the solute carrier and ATPase binding cassette families that transport drug molecules in the kidney have broad substrate specificity, there is a need to identify clinically important transporter mediated drug–drug interactions that may result in nephrotoxicity. To address this, efforts have been made to elucidate the mechanisms of drug–drug interactions and toxicity and better understand renal drug transport. The importance of transporters in the kidney has led regulatory agencies around the world to mandate drug–drug interaction and nephrotoxicity safety studies for new molecular entities that have substantial renal elimination. This review summarises the key data on the identification and characterisation of transporters found in the proximal tubule of the kidney. Differences and similarities in transporter expression and function between human and rodent species are also discussed. In addition, current renal in vitro models are explored, along with recent developments in this area.
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- 2016
18. Abundance of Drug Transporters in the Human Kidney Cortex as Quantified by Quantitative Targeted Proteomics
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Jashvant D. Unadkat, Kate M Johnson, Jonathan Himmelfarb, Caroline A. Lee, Sarah Billington, Bhagwat Prasad, Colin D.A. Brown, Git Chung, and Edward J. Kelly
- Subjects
0301 basic medicine ,Adult ,Male ,Proteomics ,Kidney Cortex ,Organic anion transporter 1 ,Abcg2 ,Pharmaceutical Science ,030226 pharmacology & pharmacy ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,medicine ,Humans ,Pharmacology ,Kidney ,biology ,Multidrug resistance-associated protein 2 ,Cell Membrane ,Membrane Proteins ,Membrane Transport Proteins ,Transporter ,Biological Transport ,Articles ,Trypsin ,Molecular biology ,030104 developmental biology ,medicine.anatomical_structure ,Membrane protein ,Pharmaceutical Preparations ,biology.protein ,Female ,medicine.drug - Abstract
Protein expression of renal uptake and efflux transporters was quantified by quantitative targeted proteomics using the surrogate peptide approach. Renal uptake transporters assessed in this study included organic anion transporters (OAT1–OAT4), organic cation transporter 2 (OCT2), organic/carnitine cation transporters (OCTN1 and OCTN2), and sodium-glucose transporter 2 (SGLT2); efflux transporters included P-glycoprotein, breast cancer resistance protein, multidrug resistance proteins (MRP2 and MRP4), and multidrug and toxin extrusion proteins (MATE1 and MATE2-K). Total membrane was isolated from the cortex of human kidneys (N = 41). The isolated membranes were digested by trypsin and the digest was subjected to liquid chromatography–tandem mass spectrometry analysis. The mean expression of surrogate peptides was as follows (given with the standard deviation, in picomoles per milligram of total membrane protein): OAT1 (5.3 ± 1.9), OAT2 (0.9 ± 0.3), OAT3 (3.5 ± 1.6), OAT4 (0.5 ± 0.2), OCT2 (7.4 ± 2.8), OCTN1 (1.3 ± 0.6), OCTN2 (0.6 ± 0.2), P-glycoprotein (2.1 ± 0.8), MRP2 (1.4 ± 0.6), MRP4 (0.9 ± 0.6), MATE1 (5.1 ± 2.3), and SGLT2 (3.7 ± 1.8). Breast cancer resistance protein (BCRP) and MATE2-K proteins were detectable but were below the lower limit of quantification. Interestingly, the protein expression of OAT1 and OAT3 was significantly correlated (r > 0.8). A significant correlation was also observed between expression of multiple other drug transporters, such as OATs/OCT2 or OCTN1/OCTN2, and SGLT2/OCTNs, OCT, OATs, and MRP2. These renal transporter data should be useful in deriving in vitro to in vivo scaling factors to accurately predict renal clearance and kidney epithelial cell exposure to drugs or their metabolites.
- Published
- 2016
19. The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule
- Author
-
Ellen van Loon, Abigail M. Dalzell, Nur Salwani Bakar, Colin D.A. Brown, Git Chung, and Sarah Jenkinson
- Subjects
Monocarboxylic Acid Transporters ,ATP Binding Cassette Transporter, Subfamily B ,Kidney Cortex ,Physiology ,Renal cortex ,Clinical Biochemistry ,OATP4C1 ,ATP-binding cassette transporter ,Cell Line ,Kidney Tubules, Proximal ,Physiology (medical) ,Cyclosporin a ,medicine ,ATP Binding Cassette Transporter, Subfamily G, Member 2 ,Humans ,RNA, Messenger ,Symporters ,biology ,Membrane Transport Proteins ,Biological Transport ,Epithelial Cells ,Transporter ,Apical membrane ,Neoplasm Proteins ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,Cell culture ,biology.protein ,ATP-Binding Cassette Transporters ,Efflux - Abstract
Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates DL-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.
- Published
- 2012
20. Human primary proximal tubule cell monolayers as a novel predictive model of nephrotoxicity
- Author
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R.D. Wilde, S. Billington, Git Chung, and Colin D.A. Brown
- Subjects
Primary (chemistry) ,medicine.anatomical_structure ,Chemistry ,Cell ,medicine ,Proximal tubule ,General Medicine ,Toxicology ,Nephrotoxicity ,Cell biology - Published
- 2016
21. Regulation of ABC transporters by hypoxia in HK‐2 human proximal tubule epithelial cells
- Author
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Colin D.A. Brown and Git Chung
- Subjects
medicine.anatomical_structure ,Chemistry ,Genetics ,medicine ,ATP-binding cassette transporter ,Proximal tubule ,Hypoxia (medical) ,medicine.symptom ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2012
22. Physiology@Newcastle
- Author
-
Megan Webster, Nichola Conlon, and Git Chung
- Published
- 2012
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