A reliable deep-learning-based method for alveolar bone quantification using a murine model of periodontitis and micro-computed tomography imaging.

OBJECTIVES: This study focuses on artificial intelligence (AI)-assisted analysis of alveolar bone for periodontitis in a mouse model with the aim to create an automatic deep-learning segmentation model that enables researchers to easily examine alveolar bone from micro-computed tomography (µCT) data without needing prior machine learning knowledge. METHODS: Ligature-induced experimental periodontitis was produced by placing a small-diameter silk sling ligature around the left maxillary second molar. At 4, 7, 9, or 14 days, the maxillary bone was harvested and processed with a µCT scanner (µCT-45, Scanco). Using Dragonfly (v2021.3), we developed a 3D deep learning model based on the U-Net AI deep learning engine for segmenting materials in complex images to measure alveolar bone volume (BV) and bone mineral density (BMD) while excluding the teeth from the measurements. RESULTS: This model generates 3D segmentation output for a selected region of interest with over 98% accuracy on different formats of µCT data. BV on the ligature side gradually decreased from 0.87 mm3 to 0.50 mm3 on day 9 and then increased to 0.63 mm3 on day 14. The ligature side lost 4.6% of BMD on day 4, 9.6% on day 7, 17.7% on day 9, and 21.1% on day 14. CONCLUSIONS: This study developed an AI model that can be downloaded and easily applied, allowing researchers to assess metrics including BV, BMD, and trabecular bone thickness, while excluding teeth from the measurements of mouse alveolar bone. CLINICAL SIGNIFICANCE: This work offers an innovative, user-friendly automatic segmentation model that is fast, accurate, and reliable, demonstrating new potential uses of artificial intelligence (AI) in dentistry with great potential in diagnosing, treating, and prognosis of oral diseases.

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.

The Nasal Solitary Chemosensory Cell Signaling Pathway Triggers Mouse Avoidance Behavior to Inhaled Nebulized Irritants.

The nasal epithelium houses a population of solitary chemosensory cells (SCCs). SCCs express bitter taste receptors and taste transduction signaling components and are innervated by peptidergic trigeminal polymodal nociceptive nerve fibers. Thus, nasal SCCs respond to bitter compounds, including bacterial metabolites, and these reactions evoke protective respiratory reflexes and innate immune and inflammatory responses. We tested whether SCCs are implicated in aversive behavior to specific inhaled nebulized irritants using a custom-built dual-chamber forced-choice device. The behavior of mice was recorded and analyzed for the time spent in each chamber. Wild-type (WT) mice exhibited an aversion to 10 mm denatonium benzoate (Den) or cycloheximide and spent more time in the control (saline) chamber. The SCC-pathway knock-out (KO) mice did not exhibit such an aversion response. The bitter avoidance behavior of WT mice was positively correlated with the concentration increase of Den and the number of exposures. Bitter-ageusic P2X2/3 double KO mice similarly showed an avoidance response to nebulized Den, excluding the taste system's involvement and pointing to an SCC-mediated major contributor to the aversive response. Interestingly, SCC-pathway KO mice showed an attraction to higher Den concentrations; however, chemical ablation of the olfactory epithelium eliminated this attraction attributed to the smell of Den. These results demonstrate that activation of SCCs leads to a rapid aversive response to certain classes of irritants with olfaction, but not gustation, contributing to the avoidance behavior during subsequent irritant exposures. This SCC-mediated avoidance behavior represents an important defense mechanism against the inhalation of noxious chemicals.

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.

The Effects of Nonnutritive Sweeteners on the Cariogenic Potential of Oral Microbiome.

Nonnutritive sweeteners (NNSs) are sugar substitutes widely used to reduce the negative health effects of excessive sugar consumption. Dental caries, one of the most prevalent chronic diseases globally, results from a pathogenic biofilm with microecological imbalance and frequent exposure to sugars. Some research has shown that certain NNSs possess less cariogenic potential than sucrose, indicating their putative effect on oral microbiome. To uncover the alterations of acidogenic pathogens and alkali-generating commensals, as well as the biofilm cariogenic potential under the influence of NNSs, we selected four common NNSs (acesulfame-K, aspartame, saccharin, and sucralose) and established single-, dual-, and multispecies in vitro culture model to assess their effects on Streptococcus mutans (S. mutans) and/or Streptococcus sanguinis (S. sanguinis) compared to sucrose with the same sweetness. The results showed that NNSs significantly suppressed the planktonic growth, acid production, and biofilm formation of S. mutans or S. sanguinis compared with sucrose in single-species cultures. Additionally, decreased S. mutans/S. sanguinis ratio, less EPS generation, and higher pH value were observed in dual-species and saliva-derived multispecies biofilms with supplementary NNSs. Collectively, this study demonstrates that NNSs inhibit the cariogenic potential of biofilms by maintaining microbial equilibrium, thus having a promising prospect as anticaries agents.

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.

TAS2R16 Activation Suppresses LPS-Induced Cytokine Expression in Human Gingival Fibroblasts.

Sustained and non-resolved inflammation is a characteristic of periodontitis. Upon acute inflammation, gingival fibroblasts release cytokines to recruit immune cells to counter environmental stimuli. The intricate regulation of pro-inflammatory signaling pathways, such as NF-κB, is necessary to maintain periodontal homeostasis. Nonetheless, how inflammation is resolved has not yet been elucidated. In this study, 22 subtypes of taste receptor family 2 (TAS2Rs), as well as the downstream machineries of Gα-gustducin and phospholipase C-ß2 (PLCß2), were identified in human gingival fibroblasts (HGFs). Various bitter agonists could induce an intensive cytosolic Ca2+ response in HGFs. More importantly, TAS2R16 was expressed at a relatively high level, and its agonist, salicin, showed robust Ca2+ evocative effects in HGFs. Activation of TAS2R16 signaling by salicin inhibited the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, at least in part, by repressing LPS-induced intracellular cAMP elevation and NF-κB p65 nuclear translocation in HGFs. These findings indicate that TAS2Rs activation in HGFs may mediate endogenous pro-inflammation resolution by antagonizing NF-κB signaling, providing a novel paradigm and treatment target for the better management of periodontitis.

Comparative analysis of the oral microbiota between iron-deficiency anaemia (IDA) patients and healthy individuals by high-throughput sequencing.

BACKGROUND: The relationship between oral microbiota and IE (infective endocarditis) is well established. Opportunistic pathogens in normal oral flora enter the bloodstream through daily oral cleaning or invasive dental procedures, leading to the occurrence of infective endocarditis. An in vitro iron-deficient condition leads to a drastic community shift in oral microbiota with increasing proportions of taxa related to infective endocarditis. To investigate the relationship among insufficient iron supply, oral microbiota and the risk of IE and to conduct a population amplification study, iron-deficiency anaemia is used as an in vivo model. METHODS: This cross-sectional study enrolled 24 primary iron-deficiency anemia (IDA) patients from 2015.6 to 2016.6 from the hematology department of West China Hospital, Sichuan University, and 24 healthy controls. High-throughput sequencing compared the dental plaque microbiota of 24 IDA (iron-deficiency anaemia) patients and 24 healthy controls. RESULTS: Sequences were classified into 12 phyla, 28 classes, 50 orders, 161 genera and 497 OTUs (the IDA and control groups shared the same 384 OTUs). Iron deficiency leads to lower internal diversity in the oral flora. The abundances of genera Corynebacterium, Neisseria, Cardiobacterium, Capnocytophaga, and Aggregatibacter were significantly higher in healthy controls, while genera Lactococcus, Enterococcus, Lactobacillus, Pseudomonas and Moraxella showed higher proportions in the IDA group (P < 0.05). The relative abundances of genera Lactococcus, Enterococcus, Pseudomonas and Moraxella were significantly negatively correlated with the concentration of serum ferritin (P < 0.05). CONCLUSIONS: Without an increase of oral streptococci, the main pathogen of IE, it is difficult to determine whether IDA can increase the risk of IE. However, the iron-deficient condition did lead to changes in the oral microbiota community structure. The genera that showed higher proportions in the IDA group were frequently reported as antibiotic-resistant. As antibiotics are commonly recommended to prevent IE before dental procedures, this study offers new ideas of personalized prevention of IE.