A5070710264- Cystic Fibrosis Research Advances
- Neonatal Respiratory Health Research
- Ion Transport and Channel Regulation
- Advanced biosensing and bioanalysis techniques
- Gastrointestinal motility and disorders
- Tracheal and airway disorders
- Cellular transport and secretion
- Advanced Fluorescence Microscopy Techniques
- Asthma and respiratory diseases
- Ion channel regulation and function
- Biotin and Related Studies
- Bacterial Genetics and Biotechnology
- Mitochondrial Function and Pathology
- Lipid Membrane Structure and Behavior
- Cancer Research and Treatments
- Legume Nitrogen Fixing Symbiosis
- Metabolism and Genetic Disorders
- Advanced Biosensing Techniques and Applications
- Digestive system and related health
- Enzyme Structure and Function
- Helicobacter pylori-related gastroenterology studies
- Neuroscience and Neuropharmacology Research
- Electrolyte and hormonal disorders
- Inhalation and Respiratory Drug Delivery
- bioluminescence and chemiluminescence research
University of California, San Francisco
2012-2024
Universidad Católica de Santa Fe
2018-2019
The University of Queensland
2011
University of Cambridge
1997-2005
Cardiovascular Institute Hospital
2000-2004
The diffusion of DNA in cytoplasm is thought to be an important determinant the efficacy gene delivery and antisense therapy. We have measured translational fluorescein-labeled double-stranded fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into nucleus HeLa cells. Diffusion was by spot photobleaching using a focused argon laser (488 nm). In aqueous solutions, coefficients water (<i>D<sub>w</sub></i>) decreased from 53 × 10<sup>−8</sup> 0.81...
The green fluorescent protein YFP‐H148Q is sensitive to halides by a mechanism involving halide binding and shift in p K . However, limitation of its low sensitivity, with d >100 mM for Cl − Indicators improved sensitivities are needed cell transport studies, particularly drug discovery high‐throughput screening, measurement concentration subcellular organelles. libraries were generated which pairs residues the vicinity site randomly mutated. An automated procedure was developed screen...
We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations purified recombinant yellow protein YFP-H148Q indicated pK(a) 7.14 in absence Cl(-), which increased to 7.86 at 150 mM Cl(-). At pH 7.5, fluorescence decreased maximally by approximately 2-fold with K(D) 100 had lifetime 3.1 ns that was independent and [Cl(-)]. Circular dichroism absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating...
Molecular diffusion in the brain extracellular space (ECS) is an important determinant of neural function. We developed a surface photobleaching method to measure fluorescently labeled macromolecules ECS cerebral cortex. The mouse was by exposure intact dura fluorescein-dextrans ( M r 4, 70, and 500 kDa). Fluorescein-dextran diffusion, detected fluorescence recovery after laser-induced cortical using confocal optics, slowed approximately threefold relative solution. Cytotoxic edema (produced...
Abstract Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of 1,2 , but limited presence divergent biology proximal trachea mice has prevented use traditional transgenic models elucidate ionocyte airway. Here we describe creation conditional genetic...
Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause fibrosis. To investigate interactions of CFTR living cells, we measured the diffusion quantum dot-labeled molecules by single particle tracking. In multiple cell lines, including airway epithelia, diffused little plasma membrane, generally not moving beyond 100-200 nm. However, became mobile over micrometer distances after 1) truncations carboxy terminus, which contains...
It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. Bindokas, Palfrey, H. and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether CFTR chloride channel a generalized pathway entry into mutations could contribute to macrophage dysfunction. The pH mature...
Background & AimsConstipation is a common clinical problem that negatively impacts quality of life and associated with significant health care costs. Activation the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel primary pathway drives fluid secretion in intestine, which maintains lubrication luminal contents. We hypothesized direct activation CFTR would cause reverse excessive dehydration stool found constipation.MethodsA cell-based, high-throughput screen was...
BackgroundCurrent modulator therapies for some cystic fibrosis-causing CFTR mutants, including N1303K, have limited efficacy. We provide evidence here to support combination potentiator (co-potentiator) therapy mutant CFTRs that are poorly responsive single potentiators.MethodsFunctional synergy screens done on N1303K and W1282X CFTR, in which small molecules were tested with VX-770, identified arylsulfonamide-pyrrolopyridine, phenoxy-benzimidazole flavone co-potentiators.ResultsA previously...
Abstract Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced concept of ‘co-potentiators’ (combination-potentiators) to rescue function some minimal mutants. Herein, a screen ~120,000 drug-like synthetic small molecules identified active co-potentiators pyrazoloquinoline, piperidine-pyridoindole,...
Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has difficult to define the role of CFTR gland fluid secretion because concerns interpreting experiments on diseased CF human airways subjected chronic infection inflammation. Here, we test by using selective inhibitor (CFTRinh-172) pig airways. Measurements single-gland rates showed inhibition both cholinergic cAMP-stimulated CFTRinh-172. Secreted...
Pendrin (SLC26A4) is a Cl(-)/anion exchanger expressed in the epithelium of inflamed airways where it thought to facilitate Cl(-) absorption and HCO3 (-) secretion. Studies using pendrin knockout mice airway epithelial cells from hearing-impaired subjects with loss function suggest involvement inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation surface liquid (ASL) volume. Here we identified small-molecule inhibitors demonstrated their efficacy increasing ASL A...
SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. loss function humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors screen 50,000 synthetic small molecules done Fischer rat thyroid (FRT) cells coexpressing and genetically encoded halide sensor. Structure-activity relationship studies were on most potent inhibitor classes screen:...
Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic transmembrane conductance regulator (CFTR) mutated in CF, and we hypothesized dysfunctional CFTR platelets, which are key participants immune responses, a central determinant of CF inflammation. We found deletion platelets produced exaggerated acute inflammation platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. loss function...
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel expressed at the apical plasma membrane. It has been proposed that C-terminal PDZ binding motif of CFTR required for its membrane targeting and PDZ-domain interactions may tether to actin cytoskeleton via soluble proteins including EBP50/NHERF1 ezrin. We measured diffusional mobility human in Madin-Darby canine kidney cells by photobleaching green fluorescent (GFP)-CFTR chimeras. After...
It has been proposed that defective submucosal gland function in CF airways is a major determinant of airway disease. We tested the hypothesis early subjects with minimal clinical Functional assays fluid secretion rate and viscosity were performed on freshly obtained nasal biopsies from 6 5 non-CF controls (age range 2-22 years). Secretions individual glands visualized by light/fluorescence microscopy after orienting immobilizing biopsy specimens custom chamber. The secreted pilocarpine,...
The mechanisms remain uncertain by which mutations in CFTR cause lung disease cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide CF lungs, was proposed to result from defective lysosomal acidification airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. Barr, M. L., von Kürthy, G., Schmid, K. W.,...
Pendrin is a Cl-/HCO3- exchanger expressed in type B and non-A, non-B intercalated cells the distal nephron, where it facilitates Cl- absorption involved Na+ acid-base balance. Pendrin-knockout mice show no fluid-electrolyte abnormalities under baseline conditions, although with double knockout of pendrin Na+/Cl- cotransporter (NCC) manifest profound salt wasting. Thus, may attenuate diuretic-induced loss, but this function remains unconfirmed. To clarify physiologic role conditions not...
Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) bone morphogenic protein antagonist Noggin. In contrast previous reports, our produces differentiated...
It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching measure diffusional mobilities of chimeras consisting green fluorescent protein (GFP) fused C terminus four enzymes: malate dehydrogenase, citrate synthase, isocitrate succinyl-CoA synthetase. The GFP-enzyme were localized...