RESUMEN
The purpose of the study was to characterize natural ventilation in full-scale gravity collection system components while measuring other parameters related to ventilation. Experiments were completed at four different locations in the wastewater collection systems of Los Angeles County Sanitation Districts, Los Angeles, California, and the King County Wastewater Treatment District, Seattle, Washington. The subject components were concrete gravity pipes ranging in diameter from 0.8 to 2.4 m (33 to 96 in.). Air velocity was measured in each pipe using a carbon-monoxide pulse tracer method. Air velocity was measured entering or exiting the components at vents using a standpipe and hotwire anemometer arrangement. Ambient wind speed, temperature, and relative humidity; headspace temperature and relative humidity; and wastewater flow and temperature were measured. The field experiments resulted in a large database of measured ventilation and related parameters characterizing ventilation in full-scale gravity sewers. Measured ventilation rates ranged from 23 to 840 L/s. The experimental data was used to evaluate existing ventilation models. Three models that were based upon empirical extrapolation, computational fluid dynamics, and thermodynamics, respectively, were evaluated based on predictive accuracy compared to the measured data. Strengths and weaknesses in each model were found and these observations were used to propose a concept for an improved ventilation model.
Asunto(s)
Contaminantes Atmosféricos/análisis , Ventilación , Eliminación de Residuos Líquidos , Movimientos del Aire , Los Angeles , Modelos Teóricos , Temperatura , Washingtón , Purificación del AguaRESUMEN
Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Células 3T3 , Animales , Biotinilación , Línea Celular , Células Cultivadas , Cloruros/metabolismo , Cresoles/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Activación del Canal Iónico , Ratones , Pirazoles/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Glándula Tiroides/fisiologíaRESUMEN
BACKGROUND: Flow cytometry of gene expression in living cells requires accurate, sensitive, nontoxic fluorescent indicators capable of detecting transcription of specific genes. This is typically achieved by using genes that encode fluorescent proteins or enzymes coupled to promoters of interest. The most commonly used reporters are green fluorescent protein and beta-galactosidase (lacZ). In this study, we characterized the performance of a cell-permeant, ratiometric, beta-lactamase substrate, coumarin cephalosporin fluorescein (CCF2/AM). We compared its characteristics with that of the beta-galactosidase/fluorescein di-beta-D-galactopyranoside reporter system. METHODS: Jurkat cell lines were generated for beta-lactamase and beta-galactosidase reporters with the use of similar plasmid constructs. Rare event flow cytometric detection for the beta-galactosidase and beta-lactamase reporters were assayed by using mixed populations of negative (WT) and positive (constitutively expressing) cells for each reporter. To determine sensitivity at low reporter copy number, we measured the activity of an unstimulated inducible promoter and detected positive events as a function of substrate incubation time. Technical issues related to data processing and optical configuration are also presented. RESULTS: The low population coefficients of variation afforded by ratiometric detection of the beta-lactamase system improved the statistical performance of the assay in comparison with a single-dye, intensity-based assay, leading to markedly improved detection for low copy number and rare events. At low levels of gene expression, beta-lactamase was detected with approximately 10-fold higher confidence than was beta-galactosidase. In rare event detection experiments, cells expressing high levels of beta-lactamase proteins were reliably detected at frequencies of 1:10(6) compared with about 1:10(4) for beta-galactosidase. CONCLUSION: The ratiometric fluorescence readout of the beta-lactamase system based on fluorescence resonance energy transfer allowed more sensitive and accurate detection of gene expression than the currently available beta-galactosidase substrates. Further, the cell-permeant nature of the substrate improved experimental convenience. These properties facilitated cell engineering and enabled a variety of applications including selection of rare cells from large populations and measurement of low-expressing or downregulated genes.