We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.cellular assays ͉ G protein-coupled receptor ͉ protein interaction A ll cells have evolved mechanisms to respond to rapid changes in the environment. Extracellular signals are detected by transmembrane receptors that translate binding into intracellular signaling events. Most signaling systems that respond to environmental cues exhibit adaptation mechanisms that afford the cell a facile response to rapid changes in their surroundings. Mechanisms to assure the rapid but transient response to environmental cues are of obvious advantage to the cell but seriously limit most assays for receptor function. We have genetically modified receptors such that transient responses to ligand result in the stable transcription of a reporter gene. The transformation of a transient intracellular response to a stable amplifiable readout provides a sensitive and quantitative assay for receptor function.We have developed an assay for receptor activation and more generally for protein-protein interaction that involves the fusion of a membrane receptor with a transcriptional activator. The membrane-bound receptor and transcription factor sequences are separated by a cleavage site for a highly specific viral protease. A second gene encodes a fusion of the viral protease with a cellular protein that interacts only with activated receptor. Ligand binding to the receptor will stimulate this proteinprotein interaction, recruiting the protease to its cleavage site. Site-specific cleavage will release the transcriptional regulator that can now enter the nucleus and activate reporter genes. Recently, a similar principle, based on the complementation of split tobacco etch virus (TEV) protease fragments, has been used to monitor protein interactions (1). Our experimental scheme derives conceptually from the mechanism of action of the Notch receptor in which ligand binding elicits proteolytic cleavage events in the receptor to release a Notch intracellular domain that translocates to the nucleus and modulates transcription of downstream target genes (2, 3) (Fig. 1A).The assay we have developed relies solely on exogenous genes in...
Sensory characteristics are important for the acceptance of thickened liquids, but those of liquids thickened to the new standards put forth by the International Dysphagia Diet Standardization Initiative (IDDSI) are unknown. This research sought to identify and rate the perception of important sensory properties of liquids thickened to levels specified in the IDDSI framework. Samples were made with water, with and without added barium sulfate, and were thickened with a cornstarch or xanthan gum based thickener. Samples were characterized using projective mapping/ultra-flash profiling to identify important sample attributes, and then with trained descriptive analysis panels to characterize those attributes in non-barium and barium thickened liquids. Three main groups of attributes were observed. Taste and flavor attributes decreased in intensity with increasing thickener. Thickener specific attributes included graininess and chalkiness for the cornstarch thickened samples, and slipperiness for the xanthan gum samples. Within the same type of thickener, ratings of thickness-related attributes (perceived viscosity, adhesiveness, manipulation, and swallowing) at different IDDSI levels were significantly different from each other. However, in non-barium samples, cornstarch samples were perceived as thicker than xanthan gum samples even though they had similar apparent viscosities at 50 s. On the other hand, the two thickeners had similar perceived thickness in the barium samples even though the apparent viscosities of cornstarch samples were higher than those of the xanthan gum samples. In conclusion, IDDSI levels can be distinguished based on sensory properties, but these properties may be affected by the type of thickener and medium being thickened.
For people with dysphagia, or difficulty swallowing, thickened liquids are used to slow bolus flow to make them easier to control. For these liquids, the oral shear rate of 50 s−1 has been adopted as the standard at which viscosity measurements are taken. However, there is evidence to suggest that other shear rates may be more appropriate to model the processes in the mouth and throat. This research compared the sensory and rheological properties of xanthan gum, guar gum, and carboxymethyl cellulose thickened liquids that had been matched for apparent viscosity at 50 s−1 to assess the validity of the current shear rate standard. Properties of gums were observed at various viscosity levels based on the International Dysphagia Diet Standardisation Initiative (IDDSI) framework. Textural sensory characteristics of samples were quantified using magnitude estimation scaling and a trained descriptive panel, while rheological measurements were taken at shear rates of 1 to 1000 s−1. Perceived slipperiness of the gums was found to be driven by thickness level at low viscosity levels, but affected by the shear thinning behavior of the gums at higher viscosity levels. Although the liquids had been matched for apparent viscosity at 50 s−1, panelists could distinguish both the perceived viscosities of the gums and their ease of swallowing, suggesting that 50 s−1 is neither appropriate to model the oral nor pharyngeal shear rates. A single oral shear rate could not be predicted from the data, and it is proposed that panelists evaluated oral viscosity using different methods at different viscosity levels. Based on the sensory data, the pharyngeal shear rate during swallowing appears to lie above 50 s−1.
Purpose During swallowing, the tongue generates the primary propulsive forces that transport material through the oral cavity toward the pharynx. Previous literature suggests that higher tongue pressure amplitudes are generated for extremely thick liquids compared with thin liquids. The purpose of this study was to collect detailed information about the modulation of tongue pressure amplitude and timing across the range from thin to moderately thick liquids. Method Tongue pressure patterns were measured in 38 healthy adults (aged under 60 years) during swallowing with 4 levels of progressively thicker liquid consistency (International Dysphagia Diet Standardisation Initiative, Levels 0 = thin , 1 = slightly thick , 2 = mildly thick , and 3 = moderately thick ). Stimuli with matching gravity flow (measured using the International Dysphagia Diet Standardisation Initiative Flow Test; Cichero et al., 2017 ; Hanson, 2016 ) were prepared both with/without barium (20% weight per volume concentration) and thickened with starch and xanthan gum thickeners. Results After controlling for variations in sip volume, thicker liquids were found to elicit significantly higher amplitudes of peak tongue pressure and a pattern of higher (i.e., steeper) pressure rise and decay slopes (change in pressure per unit time). Explorations across stimuli with similar flow but prepared with different thickeners and with/without barium revealed very few differences in tongue pressure, with the exception of significantly higher pressure amplitudes and rise slopes for nonbarium, starch-thickened slightly and mildly thick liquids. Conclusions There was no evidence that the addition of barium led to systematic differences in tongue pressure parameters across liquids with closely matched gravity flow. Additionally, no significant differences in tongue pressure parameters were found across thickening agents. Supplemental Material https://doi.org/10.23641/asha.7616537
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