IntroductionLoss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.MethodsWe investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI). Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.ResultsNanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.ConclusionNanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.
Immobilization of functional proteins such as enzymes on solid surfaces produces a variety of effects ranging from the reversal and strong inhibition to the enhancement of protein stability and function. Such effects are protein-dependent and are affected by the physical and chemical properties of the surfaces. Functional consequences of protein immobilization on the surface of gold nanoparticles (AuNPs) are protein-dependent and require thorough investigation using suitable functional tests. However, traditional approaches to making control samples, i.e., immobilized protein vs. protein in solution in absence of any nanoparticles do not provide sufficiently identical reaction conditions and complicate interpretation of the results. This report provides advice and methods for preparing AuNP-conjugated preparations generally suitable for studying the effects of immobilization on the activity and stability of different functional proteins. We use bovine catalase to illustrate our approach, but the methods are easily adaptable to any other enzyme or protein. The AuNP-immobilized enzyme showed increased stability at elevated temperatures compared to the same enzyme in solution.
Ciliated lung epithelial cells and the airway surface liquid (ASL) comprise one of the body's most important protective systems. This system is finely tuned, and perturbations to ASL rheology, ASL depth, ASL pH, the transepithelial potential, and the cilia beat frequency are all associated with disease pathology. Further, these apparently distinct properties interact with each other in a complex manner. For example, changes in ASL rheology can result from altered mucin secretion, changes in ASL pH, or changes in ASL depth. Thus, one of the great challenges in trying to understand airway pathology is that the properties of the ASL/epithelial cell system need to be assessed nearsimultaneously and without perturbing the sample. Here, we show that nanosensor probes mounted on a scanning ion conductance microscope make this possible for the first time, without any need for labeling. We also demonstrate that ASL from senescenceretarded human bronchial epithelial cells retains its native properties. Our results demonstrate that by using a nanosensor approach, it is possible to pursue faster, more accurate, more coherent, and more informative studies of ASL and airway epithelia in health and disease.
We have determined the patterns for age-specific changes related to activity of aspartateaminotransferase, alanineaminotransferase, gamma glutamyltransferase, αamylase, alkaline phosphatase and acid phosphatase in different parts of the stomach, liver, pancreas, duodenal, empty, ileac, cecum, colic and straight intestine of large white pigs at the age of 1, 7, 14, 21, 28, 60, 120 and 180 days that have been grown up in a pig-breeding farm. We have defined the principles and strength of age-specific changes related to activity of the studied ferments at the colostric, colostric-dairy (at the first transitional), first dairy, second dairy, third dairy, dairy-definitive (at the second transitional), first definitive and second definitive feeding stages in the early postnatal period of the pig systemogenesis. We have found out that in each separate feeding stage the activity of the studied ferment in various parts of the digestive organ is different. The speed of the metabolic process in the digestive system tissues, the intensity of their maturation, and functioning of each part of the digestive organ at each feeding stage are quite different. The degree of structural and chemical changes in pigs is high at early stages of the postnatal period within the first four months of pig life. They are particularly distinctive at the transitional feeding stages. The terms of particular ferment activity stabilization in digestive organs have been determined. They can be indicated at the late stages of pig feeding.
This article describes nature and intensity of agerelated changes in the activity of transferases, phosphatases and a-amylase in the tissues of middle and bung guts of the large intestine of piglets in colostrum, colostrum-dairy, first, second and third dairy, dairy-plant, first and second plant phases of nutrition in the early postnatal period of systemogenesis. Activity of enzymes is determined by the spectrophotometric method in the scientific laboratory of the Chuvash State Agricultural Academy. It was revealed that high metabolic processes in the tissues of middle and bung guts of piglets are found in the colostrum, colostrum-dairy, and the first and second phases of the definitive nutrition. The lowest activity of metabolic processes is detected in the phases of the first, second and third dairy nutrition, and the second transition phase is in between. In the tissues of external and internal middle guts, the ALT activity is stabilized from the first definitive phase. Activity of AST, GGT and a-amylase in the tissues of external and internal middle guts is not stabilized during the studied early postnatal systemogenesis. Activity of ALP and AP in tissues of these guts is stabilized from the first definitive phase. ALT, AST, GGT and aamylase do not stabilize in the bung gut tissues during the studied early postnatal systemogenesis. ALP and AP activity in tissues of this gut is stabilized from the dairy-definitive phase.
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