Statistical inference for a competing failure model based on the Wiener process and Weibull distribution.

Competing failure models with degradation phenomena and sudden failures are becoming more and more common and important in practice. In this study, the generalized pivotal quantity method was proposed to investigate the modeling of competing failure problems involving both degradation and sudden failures. In the competing failure model, the degradation failure was modeled through a Wiener process and the sudden failure was described as a Weibull distribution. For point estimation, the maximum likelihood estimations of parameters $ \mu $ and $ \sigma^2 $ were provided and the inverse estimation of parameters $ \eta $ and $ \beta $ were derived. The exact confidence intervals for parameters $ \mu $, $ \sigma^2 $, and $ \beta $ were obtained. Furthermore, the generalized confidence interval of parameter $ \eta $ was obtained through constructing the generalized pivotal quantity. Using the substitution principle, the generalized confidence intervals for the reliability function, the $ p $th percentile of lifetime, and the mean time to failure were also obtained. Simulation technique was carried out to evaluate the performance of the proposed generalized confidence intervals. The simulation results showed that the proposed generalized confidence interval was effective in terms of coverage percentage. Finally, an example was presented to illustrate the application of the proposed method.

Paxlovid mouth likely is mediated by activation of the TAS2R1 bitter receptor by nirmatrelvir.

Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has remained a public health threat since late 2019. Among the strategies rapidly developed to prevent and treat COVID-19, the antiviral medication Paxlovid (nirmatrelvir/ritonavir combination) has shown remarkable efficacy in reducing viral load and relieving clinical symptoms. Unexpectedly, a persistent bitter/bad taste, referred to as "Paxlovid mouth", has been frequently noted. Consistent with this, dysgeusia (altered taste) is listed as a main adverse effect of Paxlovid based on clinical trial data. Nirmatrelvir inhibits Mpro, a SARS-CoV-2 main protease, whereas ritonavir prolongs the activity of nirmatrelvir by slowing its metabolism. Prior usage of ritonavir in other conditions has not been linked to a persistent bad taste, despite the fact that ritonavir tastes bitter. Therefore, we hypothesized that nirmatrelvir may account for Paxlovid mouth by activating one or more of the 25 human TAS2R bitter taste receptors. Here, we show that TAS2R1 is the primary bitter receptor activated by nirmatrelvir, at concentrations as low as 15 µM, which overlaps with plasma concentrations of nirmatrelvir in a subset of patients. We also show that saccharin, a non-nutritive sweetener that may block the activity of TAS2R1, has little or no effect on nirmatrelvir-stimulated TAS2R1 activity. Such findings may help identify novel strategies to alleviate Paxlovid mouth and increase treatment compliance.

Lipopolysaccharide increases bitter taste sensitivity via epigenetic changes in Tas2r gene clusters.

T2R bitter receptors, encoded by Tas2r genes, are not only critical for bitter taste signal transduction but also important for defense against bacteria and parasites. However, little is known about whether and how Tas2r gene expression are regulated. Here, we show that in an inflammation model mimicking bacterial infection using lipopolysaccharide, the expression of many Tas2rs was significantly upregulated and mice displayed markedly increased neural and behavioral responses to bitter compounds. Using single-cell assays for transposase-accessible chromatin with sequencing (scATAC-seq), we found that the chromatin accessibility of Tas2rs was highly celltype specific and lipopolysaccharide increased the accessibility of many Tas2rs. scATAC-seq also revealed substantial chromatin remodeling in immune response genes in taste tissue stem cells, suggesting potential long-lasting effects. Together, our results suggest an epigenetic mechanism connecting inflammation, Tas2r gene regulation, and altered bitter taste, which may explain heightened bitter taste that can occur with infections and cancer treatments.

Cisplatin attenuates taste cell homeostasis and induces inflammatory activation in the circumvallate papilla.

Rationale: Gustation is important to several biological functions in mammals. However, chemotherapy drugs often harm taste perception in cancer patients, while the underlying mechanism is still unclear for most drugs and there is no effective way to restore taste function. This study investigated the effects of cisplatin on the taste cell homeostasis and gustatory function. Methods: We used both mice and taste organoid models to study the effect of cisplatin on taste buds. Gustometer assay, gustatory nerve recording, RNA-Sequencing, quantitative PCR, and immunohistochemistry was performed to analyze the cisplatin-induced alteration in taste behavior and function, transcriptome, apoptosis, cell proliferation and taste cell generation. Results: Cisplatin inhibited proliferation and promoted apoptosis in the circumvallate papilla, leading to significant impairment in taste function and receptor cell generation. The transcriptional profile of genes associated with cell cycle, metabolic process and inflammatory response was significantly altered after cisplatin treatment. Cisplatin inhibited growth, promoted apoptosis, and deferred taste receptor cell differentiation in taste organoids. LY411575, a γ-secretase inhibitor, reduced the number of apoptotic cells and increased the number of proliferative cells and taste receptor cells, potentially suggesting as a taste tissue protective agent against chemotherapy. LY411575 treatment could offset the increased number of Pax1+ or Pycr1+ cells induced by cisplatin in the circumvallate papilla and taste organoids. Conclusion: This study highlights the inhibitory effects of cisplatin on taste cell homeostasis and function, identifies critical genes and biological processes regulated by chemotherapy, and proposes potential therapeutic targets and strategy for taste dysfunction in cancer patients.

Alkaline taste sensation through the alkaliphile chloride channel in Drosophila.

The sense of taste is an important sentinel governing what should or should not be ingested by an animal, with high pH sensation playing a critical role in food selection. Here we explore the molecular identities of taste receptors detecting the basic pH of food using Drosophila melanogaster as a model. We identify a chloride channel named alkaliphile (Alka), which is both necessary and sufficient for aversive taste responses to basic food. Alka forms a high-pH-gated chloride channel and is specifically expressed in a subset of gustatory receptor neurons (GRNs). Optogenetic activation of alka-expressing GRNs is sufficient to suppress attractive feeding responses to sucrose. Conversely, inactivation of these GRNs causes severe impairments in the aversion to high pH. Altogether, our discovery of Alka as an alkaline taste receptor lays the groundwork for future research on alkaline taste sensation in other animals.

Inflammation induces bitter taste oversensitization via epigenetic changes in Tas2r gene clusters.

T2R bitter receptors, encoded by Tas2r genes, are not only critical for bitter taste signal transduction but also important for defense against bacteria and parasites. However, little is known about whether and how Tas2r gene expression are regulated. Here we show that, in an inflammation model mimicking bacterial infection, the expression of many Tas2rs are significantly up-regulated and mice displayed markedly increased neural and behavioral responses to bitter compounds. Using single-cell assays for transposase-accessible chromatin with sequencing (scATAC-seq), we found that the chromatin accessibility of Tas2rs was highly cell type specific and inflammation increased the accessibility of many Tas2rs . scATAC-seq also revealed substantial chromatin remodeling in immune response genes in taste tissue stem cells, suggesting potential long-term effects. Together, our results suggest an epigenetic mechanism connecting inflammation, Tas2r gene regulation, and altered bitter taste, which may explain heightened bitter taste that can occur with infections and cancer treatments.

Evolution of bitter receptor genes and ontogenetic dietary shift in a frog.

Vertebrate Tas2r taste receptors detect bitter compounds that are potentially poisonous. Previous studies found substantial variation in the number of Tas2r genes across vertebrates, with some frog species carrying the largest number. Peculiar among vertebrates, frogs undergo metamorphosis, often associated with a dietary shift between tadpoles and adults. A possible explanation for the large size of frog Tas2r families could be that distinct sets of Tas2r genes are required for tadpoles and adults, suggesting differential expression of Tas2r genes between tadpoles and adults. To test this hypothesis, we first examined 20 amphibian genomes and found that amphibians generally possess more Tas2r genes than do other vertebrate clades. We next focused on the American bullfrog (Lithobates catesbeianus) to examine the expression of its Tas2r genes in herbivorous tadpoles and insectivorous adult frogs. We report that close to one fifth of its 180 Tas2r genes are differentially expressed (22 genes enriched in adults and 11 in tadpoles). Tuning properties were determined for a subset of differentially expressed genes by a cell-based functional assay, with the adult-enriched Tas2r gene set covering a larger range of ligands compared to the tadpole-enriched subset. These results suggest a role of Tas2r genes in the ontogenetic dietary shift of frogs and potentially initiate a new avenue of ontogenetic analysis of diet-related genes in the animal kingdom.

Sweet taste perception in mice is blunted by PTBP1-regulated skipping of Tas1r2 exon 4.

Taste perception, initiated by activation of taste receptors in taste bud cells, is crucial for regulating nutrient intake. Genetic polymorphisms in taste receptor genes cannot fully explain the wide individual variations of taste sensitivity. Alternative splicing (AS) is a ubiquitous posttranscriptional mode of gene regulation that enriches the functional diversity of proteins. Here, we report the identification of a novel splicing variant of sweet taste receptor gene Tas1r2 (Tas1r2_∆e4) in mouse taste buds and the mechanism by which it diminishes sweet taste responses in vitro and in vivo. Skipping of Tas1r2 exon 4 in Tas1r2_∆e4 led to loss of amino acids in the extracellular Venus flytrap domain, and the truncated isoform reduced the response of sweet taste receptors (STRs) to all sweet compounds tested by generating nonfunctional T1R2/T1R3 STR heterodimers. The splicing factor PTBP1 (polypyrimidine tract-binding protein 1) promoted Tas1r2_∆e4 generation through binding to a polypyrimidine-rich splicing silencer in Tas1r2 exon 4, thus decreasing STR function and sweet taste perception in mice. Taken together, these data reveal the existence of a regulated AS event in Tas1r2 expression and its effect on sweet taste perception, providing a novel mechanism for modulating taste sensitivity at the posttranscriptional level.

Maintenance and turnover of Sox2+ adult stem cells in the gustatory epithelium.

Continuous turnover of taste bud cells in the oral cavity underlies the homeostasis of taste tissues. Previous studies have demonstrated that Sox2+ stem cells give rise to all types of epithelial cells including taste bud cells and non-gustatory epithelial cells in the oral epithelium, and Sox2 is required for generating taste bud cells. Here, we show the dynamism of single stem cells through multicolor lineage tracing analyses in Sox2-CreERT2; Rosa26-Confetti mice. In the non-gustatory epithelium, unicolored areas populated by a cluster of cells expressing the same fluorescent protein grew over time, while epithelial cells were randomly labeled with multiple fluorescent proteins by short-term tracing. Similar phenomena were observed in gustatory epithelia. These results suggest that the Sox2+ stem cell population is maintained by balancing the increase of certain stem cells with the reduction of the others. In the gustatory epithelia, many single taste buds contained cells labeled with different fluorescent proteins, indicating that a single taste bud is composed of cells derived from multiple Sox2+ stem cells. Our results reveal the characteristics of Sox2+ stem cells underlying the turnover of taste bud cells and the homeostasis of taste tissues.

Application of Two Novel Acoustic Emission Parameters on Identifying the Instability of Granite.

The issue of monitoring and early warning of rock instability has received increasing critical attention in the study of rock engineering. To investigate the damage evolution process of granite under triaxial compression tests, acoustic emission (AE) tests were performed simultaneously. This study firstly introduced two novel parameters, i.e., the coefficient of variation (CoV) of the information entropy and correlation dimension of the amplitude data from the AE tests, to identify the precursor of the failure of granite. Then the relationship between the changes in these parameters and the stress-time curve was compared and analyzed. The results of this study show that: (1) There is a strong correlation between the CoV of the information entropy and the failure process of granite. The granite failed when the CoV curve raised to a plateau, which could be used as an indicator of rock instability. (2) The fluctuation of the correlation dimension indicates the different stages during the loading process, i.e., the initial compaction stage, the linear elastic stage, the yield stage, and the failure stage. Each stage contains a descending and a rising process in the correlation dimension curve, and the exhibited starting point or the bottom point at the correlation dimension curve could be selected as the indicator point for the rock instability. (3) The combined analysis of the Information entropy and Correlation dimension can improve the accuracy of rock instability prediction. This study provides new insights into the prediction of rock instability, which has theoretical implications for the stability of subsurface engineering rock masses.

Lipopolysaccharide-induced inflammation increases nitric oxide production in taste buds.

Inducible nitric oxide synthase (iNOS) is expressed when cells are induced or stimulated by proinflammatory cytokines and/or bacterial lipopolysaccharide (LPS). iNOS is a downstream gene of the NF-κB pathway. Our previous studies demonstrated that five Nfkb genes are expressed in mouse taste epithelium and taste organoids. However, it is unclear whether activation of the NF-κB pathway could induce iNOS gene expression and increase nitric oxide (NO) production in taste buds. In this study, we investigated the expression of iNOS mRNA and protein after LPS stimulation. Our results showed that a subset of taste bud cells and taste neurons express iNOS proteins after LPS stimulation. In addition, isolated mouse taste epithelium can release NO after exposure to LPS ex vivo. In taste behavioral tests, the NO donor nitroprusside enhanced mouse aversive responses to salty, bitter, and sour taste compounds. The enhanced aversive responses were especially strong for salty taste. In conclusion, our results suggest that iNOS and NO may play a role in the inflammation-associated taste disturbances.

Expression of taste signaling elements in jejunal tissue in subjects with obesity.

Taste-signaling proteins, which are expressed throughout the digestive tract, are involved in regulating metabolism and immunity. This study aimed to determine if these genes are expressed and altered in jejunal tissues from patients with extreme obesity who received bariatric surgery. Reverse transcription polymerase chain reaction revealed that phospholipase C beta 2 and transient receptor potential channel M5 expression was downregulated in the jejunum of patients with a body mass index above 50, whereas gustducin expression remained unchanged. Our data suggest that taste-signaling dysregulation might contribute to obesity.

RNF43/ZNRF3 negatively regulates taste tissue homeostasis and positively regulates dorsal lingual epithelial tissue homeostasis.

Taste bud cells are renewed throughout life in a process requiring innervation. Recently, we reported that R-spondin substitutes for neuronal input for taste cell regeneration. R-spondin amplifies WNT signaling by interacting with stem-cell-expressed E3 ubiquitin ligases RNF43/ZNRF3 (negative regulators of WNT signaling) and G-protein-coupled receptors LGR4/5/6 (positive regulators of WNT signaling). Therefore, we hypothesized that RNF43/ZNRF3 may serve as a brake, controlled by gustatory neuron-produced R-spondin, for regulating taste tissue homeostasis. Here, we show that mice deficient for Rnf43/Znrf3 in KRT5-expressing epithelial stem/progenitor cells (RZ dKO) exhibited taste cell hyperplasia; in stark contrast, epithelial tissue on the tongue degenerated. WNT signaling blockade substantially reversed all these effects in RZ dKO mice. Furthermore, innervation becomes dispensable for taste cell renewal in RZ dKO mice. We thus demonstrate important but distinct functions of RNF43/ZNRF3 in regulating taste versus lingual epithelial tissue homeostasis.

Immune Regulatory Roles of Cells Expressing Taste Signaling Elements in Nongustatory Tissues.

G protein-coupled taste receptors and their downstream signaling elements, including Gnat3 (also known as α-gustducin) and TrpM5, were first identified in taste bud cells. Subsequent studies, however, revealed that some cells in nongustatory tissues also express taste receptors and/or their signaling elements. These nongustatory-tissue-expressed taste receptors and signaling elements play important roles in a number of physiological processes, including metabolism and immune responses. Special populations of cells expressing taste signaling elements in nongustatory tissues have been described as solitary chemosensory cells (SCCs) and tuft cells, mainly based on their morphological features and their expression of taste signaling elements as a critical molecular signature. These cells are typically scattered in barrier epithelial tissues, and their functions were largely unknown until recently. Emerging evidence shows that SCCs and tuft cells play important roles in immune responses to microbes and parasites. Additionally, certain immune cells also express taste receptors or taste signaling elements, suggesting a direct link between chemosensation and immune function. In this chapter, we highlight our current understanding of the functional roles of these "taste-like" cells and taste signaling pathways in different tissues, focusing on their activities in immune regulation.

Local Adaptation of Bitter Taste and Ecological Speciation in a Wild Mammal.

Sensory systems are attractive evolutionary models to address how organisms adapt to local environments that can cause ecological speciation. However, tests of these evolutionary models have focused on visual, auditory, and olfactory senses. Here, we show local adaptation of bitter taste receptor genes in two neighboring populations of a wild mammal-the blind mole rat Spalax galili-that show ecological speciation in divergent soil environments. We found that basalt-type bitter receptors showed higher response intensity and sensitivity compared with chalk-type ones using both genetic and cell-based functional analyses. Such functional changes could help animals adapted to basalt soil select plants with less bitterness from diverse local foods, whereas a weaker reception to bitter taste may allow consumption of a greater range of plants for animals inhabiting chalk soil with a scarcity of food supply. Our study shows divergent selection on food resources through local adaptation of bitter receptors, and suggests that taste plays an important yet underappreciated role in speciation.

Up-regulation of gasdermin C in mouse small intestine is associated with lytic cell death in enterocytes in worm-induced type 2 immunity.

"Taste-like" tuft cells in the intestine trigger type 2 immunity in response to worm infection. The secretion of interleukin-13 (IL-13) from type 2 innate lymphoid cells (ILC2) represents a key step in the tuft cell-ILC2 cell-intestinal epithelial cell circuit that drives the clearance of worms from the gut via type 2 immune responses. Hallmark features of type 2 responses include tissue remodeling, such as tuft and goblet cell expansion, and villus atrophy, yet it remains unclear if additional molecular changes in the gut epithelium facilitate the clearance of worms from the gut. Using gut organoids, we demonstrated that IL-4 and IL-13, two type 2 cytokines with similar functions, not only induced the classical type 2 responses (e.g., tuft cell expansion) but also drastically up-regulated the expression of gasdermin C genes (Gsdmcs). Using an in vivo worm-induced type 2 immunity model, we confirmed the up-regulation of Gsdmcs in Nippostrongylus brasiliensis-infected wild-type C57BL/6 mice. Consistent with gasdermin family members being principal effectors of pyroptosis, overexpression of Gsdmc2 in human embryonic kidney 293 (HEK293) cells triggered pyroptosis and lytic cell death. Moreover, in intestinal organoids treated with IL-4 or IL-13, or in wild-type mice infected with N. brasiliensis, lytic cell death increased, which may account for villus atrophy observed in worm-infected mice. Thus, we propose that the up-regulated Gsdmc family may be major effectors for type 2 responses in the gut and that Gsdmc-mediated pyroptosis may provide a conduit for the release of antiparasitic factors from enterocytes to facilitate the clearance of worms.

Inhibition of Bitter Taste from Oral Tenofovir Alafenamide.

Children have difficulty swallowing capsules. Yet, when presented with liquid formulations, children often reject oral medications due to their intense bitterness. Presently, effective strategies to identify methods, reagents, and tools to block bitterness remain elusive. For a specific bitter-tasting drug, identification of the responsible bitter receptors and discovery of antagonists for those receptors can provide a method to block perceived bitterness. We have identified a compound (6-methylflavone) that can block responses to an intensely bitter-tasting anti-human immunodeficiency virus (HIV) drug, tenofovir alafenamide (TAF), using a primary human taste bud epithelial cell culture as a screening platform. Specifically, TAS2R39 and TAS2R1 are the main type 2 taste receptors responding to TAF observed via heterologously expressing specific TAS2R receptors into HEK293 cells. In this assay, 6-methylflavone blocked the responses of TAS2R39 to TAF. In human sensory testing, 8 of 16 subjects showed reduction in perceived bitterness of TAF after pretreating (or "prerinsing") with 6-methylflavone and mixing 6-methylflavone with TAF. Bitterness was completely and reliably blocked in two of these subjects. These data demonstrate that a combined approach of human taste cell culture-based screening, receptor-specific assays, and human psychophysical testing can successfully discover molecules for blocking perceived bitterness of pharmaceuticals, such as the HIV therapeutic TAF. Our hope is to use bitter taste blockers to increase medical compliance with these vital medicines. SIGNIFICANCE STATEMENT: Identification of a small molecule that inhibits bitter taste from tenofovir alafenamide may increase the compliance in treating children with human immunodeficiency virus infections.

R-spondin substitutes for neuronal input for taste cell regeneration in adult mice.

Taste bud cells regenerate throughout life. Taste bud maintenance depends on continuous replacement of senescent taste cells with new ones generated by adult taste stem cells. More than a century ago it was shown that taste buds degenerate after their innervating nerves are transected and that they are not restored until after reinnervation by distant gustatory ganglion neurons. Thus, neuronal input, likely via neuron-supplied factors, is required for generation of differentiated taste cells and taste bud maintenance. However, the identity of such a neuron-supplied niche factor(s) remains unclear. Here, by mining a published RNA-sequencing dataset of geniculate ganglion neurons and by in situ hybridization, we demonstrate that R-spondin-2, the ligand of Lgr5 and its homologs Lgr4/6 and stem-cell-expressed E3 ligases Rnf43/Znrf3, is expressed in nodose-petrosal and geniculate ganglion neurons. Using the glossopharyngeal nerve transection model, we show that systemic delivery of R-spondin via adenovirus can promote generation of differentiated taste cells despite denervation. Thus, exogenous R-spondin can substitute for neuronal input for taste bud cell replenishment and taste bud maintenance. Using taste organoid cultures, we show that R-spondin is required for generation of differentiated taste cells and that, in the absence of R-spondin in culture medium, taste bud cells are not generated ex vivo. Thus, we propose that R-spondin-2 may be the long-sought neuronal factor that acts on taste stem cells for maintaining taste tissue homeostasis.

Loss of sweet taste despite the conservation of sweet receptor genes in insectivorous bats.

The evolution of taste perception is usually associated with the ecology and dietary changes of organisms. However, the association between feeding ecology and taste receptor evolution is unclear in some lineages of vertebrate animals. One example is the sweet taste receptor gene Tas1r2 Previous analysis of partial sequences has revealed that Tas1r2 has undergone equally strong purifying selection between insectivorous and frugivorous bats. To test whether the sweet taste function is also important in bats with contrasting diets, we examined the complete coding sequences of both sweet taste receptor genes (Tas1r2 and Tas1r3) in 34 representative bat species. Although these two genes are highly conserved between frugivorous and insectivorous bats at the sequence level, our behavioral experiments revealed that an insectivorous bat (Myotis ricketti) showed no preference for natural sugars, whereas the frugivorous species (Rousettus leschenaultii) showed strong preferences for sucrose and fructose. Furthermore, while both sweet taste receptor genes are expressed in the taste tissue of insectivorous and frugivorous bats, our cell-based assays revealed striking functional divergence: the sweet taste receptors of frugivorous bats are able to respond to natural sugars whereas those of insectivorous bats are not, which is consistent with the behavioral preference tests, suggesting that functional evolution of sweet taste receptors is closely related to diet. This comprehensive study suggests that using sequence conservation alone could be misleading in inferring protein and physiological function and highlights the power of combining behavioral experiments, expression analysis, and functional assays in molecular evolutionary studies.

SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue.

Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.