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The Egyptian tortoise (Testudo kleinmanni) is remarkably adapted to its harsh desert environment, a characteristic that is crucial for its survival under extreme conditions. This study was aimed at providing a deeper understanding of the lingual salivary gland structures in the Egyptian tortoise and examining how these structures help the tortoise manage hydration and nutrition in arid conditions. Utilizing a combination of light microscopy and immunofluorescence, this research introduced pioneering methods involving seven different antibodies, marking a first in the study of reptilian salivary glands. Our investigations categorized the tortoise's salivary glands into papillary and non-papillary types. The papillary glands were further classified into superficial, deep, interpapillary, and intraepithelial salivary glands, while non-papillary glands included superficial and deep lingual types. Structurally, these glands are organized into lobules, delineated by interlobular septa, and are equipped with a duct system comprising interlobular, intercalated, and main excretory ducts with gland openings on the tongue's surface and the papillae surfaces. Notably, the superficial glands displayed both tubuloalveolar and acinar configurations, whereas the deep lingual glands were exclusively acinar. Immunofluorescence results indicated that α-smooth muscle actin (α-SMA) was prevalent in myoepithelial cells, myofibroblasts, and blood vessels, suggesting their integral role in glandular function and support. E-cadherin was predominantly found in epithelial cells, enhancing cell adhesion and integrity, which are critical for efficient saliva secretion. Importantly, Mucin 1 (MUC1) and Mucin 5B (MUC5B) staining revealed that most glands were mucous in nature, with MUC5B specifically marking mucin within secretory cells, confirming their primary function in mucous secretion. PDGFRα and CD34 highlighted the presence of telocytes and stromal cells within the glandular and interlobular septa, indicating a role in structural organization and possibly in regenerative processes. Cytokeratin 14 expression was noted in the basal cells of the glands, underscoring its role in upholding the structural foundation of the epithelial barrier. In conclusion, this detailed morphological and immunological characterization of the Egyptian tortoise's salivary glands provides new insights into their complex structure and essential functions. These findings not only enhance our understanding of reptilian physiology but also underline the critical nature of salivary glands in supporting life in arid environments. This study's innovative use of a broad range of immunofluorescence markers opens new avenues for further research into the adaptive mechanisms of reptiles., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This is the first study to describe the subtypes, number and distribution of mast cells (MC) in cat tongue by histochemical and immunohistochemical methods. Six male adult felines' tongue tissue samples consist of the study's material. Samples were fixed in 10% formaldehyde. MC number and distribution in the feline tongue were assessed using toluidine blue. Also, sections taken from blocks were stained in alcian blue/safranin O (AB/SO) combined dyes to determine the MC subtypes. The Streptavidin biotin complex method using anti-chymase and anti-tryptase primary antibodies was used for immunohistochemistry. Metachromatic MCs were mainly observed in the lamina propria close to the multilayered keratinized stratified squamous epithelium. The high number of MCs in this region may be because the dorsal surface of the tongue plays an essential role in the defence system of tongue tissue and, thus, of the body as a whole. Additionally, the number of MCs stained with AB (+) (1.7 ± 0.08) in the feline tongue was statistically higher than those with SO (+) (0.18 ± 0.02). This might be interpreted as an indication that MC heterogeneity may be due not only to their staining properties but also to their localization. It is also conceivable that the high histamine content may be a factor in this. Tryptase-positive MCs were found in the loose connective tissue around blood vessels, between the glands, as solitary cells, or in groups of several cells. Chymase-positive MCs were observed more individually rather than in groups. Moreover, chymase-positive MCs were detected to be located in the filiform papillae subepithelial and in the blood vessels' immediate vicinity. Animals often lick themselves to clean themselves and promote healing. For this reason, it is very important to protect the tongue, which is in direct contact with the external environment, against foreign agents. Considering both the functional and protective properties of the tongue, we concluded that MCs may play a role in oral cavity immunity and protective effect., (© 2024 The Author(s). Anatomia, Histologia, Embryologia published by Wiley‐VCH GmbH.)

Cattle egret (Bubulcus ibis) is a cosmopolitan heron species, with least concern conservation status. There are limited literatures on the anatomy of this bird, especially in relation to its sensory organs, hence we here investigated the gross morphological and histomorphometric features of its tongue. The tongues of twelve healthy juvenile cattle egrets were examined in situ for morphological appearance and gross morphometric measurements were determined ex situ. Routine histology was conducted on the tongue tissue with parameters such as epithelial and lamina propia heights, lingual muscle and entoglossal cartilage heights evaluated. Grossly, the tongue was divided into three parts name; apex, body and the root. It was arrow shaped, conforming to the shape of the beak, with a laryngeal mound bounded caudally by the pharyngeal papillae at its root. A massive entoglossal cartilage formed the core of the cranial apex, ventral body portion, and caudal aspect of the root. Histologically, the lingual mucosa possessed keratinized squamous epithelium in all its divisions, with spinous conical papillae being characteristic of the cranial apical mucosa. The body lingual mucosa possessed foliate papillae on the dorsal aspects, while filiform papillae were prominent in the ventral portions. The lingual root uniquely possessed numerous glandular ducts in its lamina propia as well as localized adipocytes. Overall, the regression analysis data showed that the body weight can be conveniently predicted from tongue parameters. This study has thus provided additional knowledge on the anatomy of the birds and the generated data could prove useful in comparative regional anatomy.

Little is known about the molecular mechanisms underlying drug-induced taste disorders, which can cause malnutrition and reduce quality of life. One of taste disorders is known adverse effects of bisphosphonates, which are administered as anti-osteoporotic drugs. Therefore, the present study evaluated the effects of risedronate (a bisphosphonate) on taste bud cells. Expression analyses revealed that farnesyl diphosphate synthase (FDPS, a key enzyme in the mevalonate pathway) was present in a subset of mouse taste bud and tongue epithelial cells, especially type III sour-sensitive taste cells. Other mevalonate pathway-associated molecules were also detected in mouse taste buds. Behavioral analyses revealed that mice administered risedronate exhibited a significantly enhanced aversion to HCl but not for other basic taste solutions, whereas the taste nerve responses were not affected by risedronate. Additionally, the taste buds of mice administered risedronate exhibited significantly lower mRNA expression of desmoglein-2, an integral component of desmosomes. Taken together, these findings suggest that risedronate may interact directly with FDPS to inhibit the mevalonate pathway in taste bud and tongue epithelial cells, thereby affecting the expression of desmoglein-2 related with epithelial barrier function, which may lead to alterations in behavioral responses to HCl via somatosensory nerves., (© 2022. The Author(s).)

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., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

A triangular lingual prominence (LP) is a characteristic part of the tongue in Anseriformes containing adipose tissue. The parakeratinized epithelium (PEp) covers the LP. Studies aimed to describe the histogenesis of PEp during the process of the intensive formation of the LP in domestic goose during embryonic period and to determine the structural readiness to perform a protective function. The study were conducted by using LM, SEM and TEM technique. The results revealed that on day 16th the undifferentiated epithelium of LP transformed into the typical avian multilayered epithelium. Contrary to pattern of histogenesis of parakeratinized epithelium on the lingual body, on the medial and lateral areas of the elongating and bulging LP were formed epithelial furrows. Which around 20th day, on lateral areas of LP deepened up to half of epithelium, whereas on the medial area began to fade. The ultrastructure of cells lying in furrows indicated progressive apoptosis-like degeneration. On the 25th day, shallow furrows were only present on lateral areas, where bulging of LP was continued. Whereas the epithelium on medial area started cornification by the accumulation of cytokeratin fibers. Lack of the periderm during the development of the PEp of the LP indicated its endodermal origin., (© 2021. The Author(s).)

Leptin is known to selectively suppress neural and taste cell responses to sweet compounds. The sweet suppressive effect of leptin is mediated by the leptin receptor Ob-Rb, and the ATP-gated K + (K ATP ) channel expressed in some sweet-sensitive, taste receptor family 1 member 3 (T1R3)-positive taste cells. However, the intracellular transduction pathway connecting Ob-Rb to K ATP channel remains unknown. Here we report that phosphoinositide 3-kinase (PI3K) mediates leptin's suppression of sweet responses in T1R3-positive taste cells. In in situ taste cell recording, systemically administrated leptin suppressed taste cell responses to sucrose in T1R3-positive taste cells. Such leptin's suppression of sucrose responses was impaired by co-administration of PI3K inhibitors (wortmannin or LY294002). In contrast, co-administration of signal transducer and activator of transcription 3 inhibitor (Stattic) or Src homology region 2 domain-containing phosphatase-2 inhibitor (SHP099) had no effect on leptin's suppression of sucrose responses, although signal transducer and activator of transcription 3 and Src homology region 2 domain-containing phosphatase-2 were expressed in T1R3-positive taste cells. In peeled tongue epithelium, phosphatidylinositol (3,4,5)-trisphosphate production and phosphorylation of AKT by leptin were immunohistochemically detected in some T1R3-positive taste cells but not in glutamate decarboxylase 67-positive taste cells. Leptin-induced phosphatidylinositol (3,4,5)-trisphosphate production was suppressed by LY294002. Thus, leptin suppresses sweet responses of T1R3-positive taste cells by activation of Ob-Rb-PI3K-K ATP channel pathway., (© 2020 International Society for Neurochemistry.)

Objective To investigate the expression and clinical significance of phosphatase and tension homolog deleted on chromosome ten (PTEN) and stimulator of interferon genes (STING) in human tongue squamous cell carcinoma (TSCC). Methods The expression of PTEN and STING protein in 65 pairs of TSCC and paracancerous tissues was detected by immunohistochemical EnVision method, and the relationships between PTEN, STING and clinicopathological parameters, overall survival (OS) and prognosis were analyzed by statistical methods. Results Compared with the adjacent tissues, the expression of PTEN in TSCC significantly decreased, and the expression of STING significantly increased. PTEN was negatively correlated with STING. In TSCC, the expression of PTEN and STING were correlated with pathological grade, TNM stage and lymph node metastasis. There were no significant correlations between the expression intensity of PTEN, STING and the gender and age of patients. The low expression of PTEN and the high expression of STING in TSCC tissues were significantly associated with poor prognosis and significantly shortened overall survival of patients. Conclusion TSCC patients with low expression of PTEN and high expression of STING have poor prognosis and short survival time. Combined detection of PTEN and STING expression is helpful to evaluate the degree of tumor progression and patient prognosis.

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., Competing Interests: NO authors have competing interests.

Sense of taste is central to evaluate food before digestion. Taste stem cells undergo constant differentiation throughout the life. However, the mechanism underlying the generation of taste receptor cells is still not clear. Here, we cultured taste organoids from either Lgr5+ or Lgr5-cells, and found the preferential generation of Car4+ and Gustducin + taste receptor cells in organoids derived from Lgr5+ cells in circumvallate, foliate or fungiform papillae. Taste organoids derived from Lgr5+ cells in circumvallate papillae of neonatal mice showed stronger capacity to generate taste receptor cells compared to the organoids from Lgr5+ cells of the adult circumvallate papillae. Massive transcriptional differences were found in multiple signaling pathways including taste transduction between organoids derived from circumvallate papillae of adult and neonatal mice. Inhibiting the Notch signaling pathway by LY411575 enhanced taste receptor cell generation in organoids from circumvallate papillae and modulated multiple signaling pathways. Thus, we concluded that receptor cell generation in taste organoids was age-related and regulated via multiple signaling pathways., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The herbicide dichlorophenoxyacetic acid (2,4-D) is one of the most widely used crop spraying products in the world. Some pesticides induce the degranulation of mast cells and increase allergic responses. This is the first study to evaluate the damage to the oral mucosa after an experimental simulation of environmental inhalation exposure to the 2,4-D herbicide. The aim of this study was evaluate the possible oral damage caused by acute inhalation exposure to the herbicide 2,4-D., Results: There was a difference between the exposure concentrations in relation to tissue congestion intensity (p = 0.002) and mast cell counts (p = 0.002), a difference in the evaluation of the interaction between the exposure concentrations and nebulization time in the dorsum epithelium thickness (p = 0.013), and a significant correlation between the epithelial thickness and the number of nucleoli organizing regions on the dorsum of the tongue (p = 0.048)., Conclusions: Even after acute exposure, the herbicide 2,4-D had the potential to damage the oral epithelium, especially at higher doses.

This study was conducted with the tongue samples of different life stages of hilsa, that is, adult Marine hilsa, adult Riverine hilsa, and Riverine juvenile hilsa, respectively. Three types of taste buds (Types I, II, and III based on their elevation from the epithelium at different levels) of the tongue, which may be to ensure full utilization of the gustatory ability of the fish were rocorded. Presence of specific taste buds indicate that the fish hilsa dwells in turbid waters with a possible preference toward diatom like planktonic food source. Enhanced expression of taste receptors (T1R1 and T1R3) and associated stimulatory G-proteins subunits on tongue also indicate occurrence of amino acid like substances that guided sensory cues for feeding by this fish. A firm regularity or stringency of the free surface of the epithelial cells may be attributed to compactly arranged microridges. These structures protect against physical abrasions potentially caused during food manoeuvring and swallowing. In our present observations, the surface architectures of the tongue of hilsa are discussed within the background of migratory adaptation of the species in the context of feeding and habitat preferences., (© 2020 Wiley Periodicals, Inc.)

Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include (i) the stabilization of these structures under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, (iii) and the data analysis that ranges in complexity from whole specimens to single cells. Here, we addressed these issues for the time-lapse analysis of Ca 2+ signaling in spheroids composed of human tongue-derived HTC-8 cells upon perfusion of gustatory substances. Live cell imaging setups for confocal and light sheet microscopy were developed that allow simple and robust spheroid stabilization and high-resolution microscopy with perfusion. Visualization of spheroids made of HTC-8 cells expressing the G-GECO fluorescent Ca 2+ sensor revealed Ca 2+ transients that showed similar kinetics but different amplitudes upon perfusion of bitter compounds Salicine and Saccharin. Dose-dependent responses to Saccharin required extracellular Ca 2+ . From the border towards the center of spheroids, compound-induced Ca 2+ signals were progressively delayed and decreased in amplitude. Stimulation with ATP led to strong Ca 2+ transients that were faster than those evoked by the bitter compounds and blockade of purinergic receptors with Suramin abutted the response to Saccharin, suggesting that ATP mediates a positive autocrine and paracrine feedback. Imaging of ATP-induced Ca 2+ transients with light sheet microscopy allowed acquisition over a z-depth of 100 μm without losing spatial and temporal resolution. In summary, the presented approaches permit the study of fast cellular signaling in three-dimensional cultures upon compound perfusion., Competing Interests: Declaration of Competing Interest No competing interests are declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The tayra (Eira barbara) is a mammal belonging to the Mustelidae family that occurs in all Brazilian biomes. The present work aimed to describe the morphology of the tongue of these specimens highlighting their structures and particularities that will serve as a subsidy to elucidate the anatomy of the same and for comparative studies among other species of domestic and wild animals. Five adult male specimens of E. barbara were studied, which were fixed using 10% aqueous formaldehyde solution. The tongue was removed by opening the oral cavity and separating the maxillary/mandible bone complex. Being in possession of the material, photodocumentations and collection of the fragments were made for the proper preparation of histological slides and Scanning Electron Microscopy (SEM). The lingual papillae found in tayra were mechanical: filiform and conical; and gustative: fungiform and circumvallated. Histologically, the papillae are constituted by keratinized stratified epithelium and in the innermost region, it was composed of tissue connective dense unshaped followed by a layer of muscle bundles of skeletal striated. In the region of the root of the tongue of E. barbara, there were a set of small mixed salivary glands (serous and mucous) and the punctual presence of gustatory corpuscles at the level of epithelium. The morphological description of the E. barbara tongue revealed similarity to that described in literature for other domestic and wild mammals. However, the particularity of the absence of foliate papilla and the quantitative of four papillae circumvallate in the region of the root of the tongue of this species., (© 2020 Blackwell Verlag GmbH.)

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco's modified Eagle's medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs., (© 2020 The Author(s).)

Extraocular muscles (EOMs) show resistance to muscle dystrophies and sarcopenia. It has been recently demonstrated that they are endowed with different types of myogenic cells, all of which present an outstanding regenerative potential. Neurotrophins are important modulators of myogenic regeneration and act promoting myoblast proliferation, enhancing myogenic fusion rates and protecting myotubes from inflammatory stimuli. Here, we adapted the pre-plate cell isolation technique to obtain myogenic progenitors from the rat EOMs, and quantified their in vitro expression of neurotrophins and their receptors by RT-qPCR and immunohistochemistry, respectively. The results were compared with the expression on progenitors isolated from buccinator, tongue and limb muscles. Our quantitative analysis of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) transcripts showed, for the first time, that EOMs-derived cells express more of these factors and that they expressed TrkA, but not TrkB and TrkC receptors. On the contrary, the immunofluorescence analysis demonstrated high expression of p75 NTR on all myogenic progenitors, with the EOMs-derived cells showing higher expression. Taken together, these results suggest that the intrinsic trophic differences between EOMs-derived myogenic progenitors and their counterparts from other muscles could explain why those cells show higher proliferative and fusion rates, as well as better regenerative properties., Competing Interests: Colombo, E.; Romaggi, S.; Medico, E.; Menon, R.; Mora, M.; Falcone, C.; et al. Human neurotrophin receptor p75NTR defines differentiation-oriented skeletal muscle precursor cells: implications for muscle regeneration. J. Neuropathol. Exp. Neurol. 2011, 70, 133–142.

During a foot-and-mouth disease (FMD) outbreak, transport and testing of potentially infectious samples, including epithelium from suspect lesions, presents a biosafety risk, particularly in FMD-free countries. Therefore, treatment to inactivate virus prior to transport is important. Tongue epithelium from cattle infected with FMD virus (FMDV) serotype O (O ALG/3/2014 - Lineage O/ME-SA/Ind-2001d) or A (A IRN/22/2015 - Lineage A/ASIA/G-VII) was incubated in RNAlater, RNA Shield or phosphate-buffered saline (pH 7.4) at room temperature for 2, 6, 24 or 48 h. After incubation, tissues were homogenised and tested by virus titration. Viral RNA in the homogenate was quantified by RT-qPCR, used for sequencing, and transfected into LFBK αVβ6 cells to recover infectious virus. RNAlater reduced A IRN/22/2015 titres by 4 log 10 after 24 h, and completely after 48 h incubation. While O ALG/3/2014 was detected by VI after 2, 6 and 24 h, titration yielded no infectious virus, likely as a result of freeze-thawing. RNA Shield was cytotoxic at high concentrations but was effective at inactivating both strains after 24 h. Regardless of reagent or inactivation period, RT-qPCR, VP1 sequencing, and transfection of RNA to recover infectious virus were possible. RNA Shield appears a better choice for FMDV inactivation in tissues, however 24 h incubation is recommended., (Copyright © 2019. Published by Elsevier B.V.)

In this study, we aimed to observe the localization and expression of peptide hormones-leptin, ghrelin and obestatin-in the sheep tongue by immunohistochemistry. For that purpose, tongues of ten adult sheep were used. Leptin expression of moderate intensity was observed in the basal and parabasal epithelial cells of the luminal epithelium, and leptin was strongly expressed in the taste buds of the circumvallate and fungiform papillae and in von Ebner's glands. Ghrelin was primarily expressed in some of the skeletal muscle cells and the smooth muscle cells of the middle layer of blood vessels. A strong expression was observed in the epithelial cells lining the base of the groove surrounding the circumvallate papillae. Obestatin expression was particularly strong in the epithelial cells of the salivary ducts. It was also stronger in the von Ebner's glands than in the seromucous glands. Leptin, ghrelin and obestatin were shown to be produced at varying levels in different cell types, including epithelial, stromal and skeletal muscle cells, as well as in ganglion neurons, neural plexuses and blood vessels in the sheep tongue. Cellular localization and expression of these peptide hormones have not been investigated in many species including sheep., (© 2019 Blackwell Verlag GmbH.)

Bumblebees and some other tiny animals feed on nectar by visiting flowers in their neighborhood. Some bee species appear to be highly specialized, their tongue being adapted to specific flowers. Bombus terrestris in contrast is able to feed on a wide variety of flowers and can thus be considered as a kind of universal nectar catcher. Since plant nectars show highly variable sugar content, Bombus terrestris have developed a capture mechanism that works for almost any fluid viscosity. Their tongues are decorated with very elongated papillae forming a hairy coating surrounding a rod-like main stalk. When settled on a flower, Bombus rapidly dip their tongue into the inflorescence to catch the highly sought-after nectar. To determine the physical mechanism at the origin of this outstanding ability, the capture dynamics was followed from videos recorded during viscous fluid ingestion. Surprisingly, the volume per lap and the lapping frequency are independent of the fluid viscosity over three orders of magnitude. To explain this observation, we designed a physical model of viscous dipping with structured rods. Predictions of the model compared to observations for bees showed that the nectar is not captured with the help of viscous drag, as proposed in the Landau-Levich-Derjaguin model, but thanks to the hairy structure that traps the viscous fluid, capillary forces drastically limiting the drainage. Our approach can be transposed to others nectar foragers such as bats and hummingbirds.

Objective: Accumulating evidence suggests that macrophages are involved in the immunoediting of oral squamous cell carcinoma but the role of macrophages in oral carcinogenesis is unclear. We aimed to clarify the role of macrophages in oral leukoplakia, which is the most common oral potentially malignant disorder from immunotolerance viewpoint., Materials and Methods: The study included 24 patients who underwent surgical resection for tongue leukoplakia. The relationships between macrophage markers and clinicopathological factors were assessed. Conditioned medium was harvested from the CD163 + human monocytic leukaemia cell line, THP-1. The phenotypic alteration of human oral keratinocytes by the conditioned medium treatment was assessed using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Moreover, the clinical samples were evaluated using immunohistochemistry., Results: Tongue leukoplakia tissues with high CD163 + macrophage infiltration were associated with significantly higher degrees of epithelial dysplasia, abnormal Ki-67 expression and cytokeratin13 loss when compared with the tissues with low CD163 + macrophage infiltration. In vitro, CD163 + THP-1 conditioned medium induced immunosuppressive molecules, especially interleukin-10 (IL-10) in human oral keratinocytes. The IL-10 expression levels showed significant positive correlations with not only the numbers of FOXP3 + regulatory T cells but also that of CD163 + macrophages., Conclusions: In tongue leukoplakia, CD163 + macrophages infiltration correlates with immunosuppressive cytokine IL-10 expression., Competing Interests: The authors have no conflict of interest., (©2019 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

The vertebrate tongue is a complex muscular organ situated in the oral cavity and involved in multiple functions including mastication, taste sensation, articulation and the maintenance of oral health. Although the gross embryological contributions to tongue formation have been known for many years, it is only relatively recently that the molecular pathways regulating these processes have begun to be discovered. In particular, there is now evidence that the Hedgehog, TGF-Beta, Wnt and Notch signaling pathways all play an important role in mediating appropriate signaling interactions between the epithelial, cranial neural crest and mesodermal cell populations that are required to form the tongue. In humans, a number of congenital abnormalities that affect gross morphology of the tongue have also been described, occurring in isolation or as part of a developmental syndrome, which can greatly impact on the health and well-being of affected individuals. These anomalies can range from an absence of tongue formation (aglossia) through to diminutive (microglossia), enlarged (macroglossia) or bifid tongue. Here, we present an overview of the gross anatomy and embryology of mammalian tongue development, focusing on the molecular processes underlying formation of the musculature and connective tissues within this organ. We also survey the clinical presentation of tongue anomalies seen in human populations, whilst considering their developmental and genetic etiology., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Neuronatin ( Nnat ) is expressed in the pituitary, pancreas, and other tissues; however, the function of NNAT is still unclear. Recent studies have demonstrated that NNAT is localized in the sex-determining region Y-box 2-positive stem/progenitor cells in the developing rat pituitary primordium and is downregulated during differentiation into mature hormone-producing cells. Moreover, NNAT is widely localized in subcellular organelles, excluding the Golgi. Here, we further evaluated NNAT-positive cells and intracellular localization in embryonic and postnatal rat tissues such as the pancreas, tongue, whisker hair follicle, and testis. Immunohistochemistry revealed that NNAT was localized in undifferentiated cells (i.e., epithelial basal cells and basement cells in the papillae of the tongue and round and elongated spermatids of the testis) as well as in differentiated cells (insulin-positive cells and exocrine cells of the pancreas, taste receptor cells of the fungiform papilla, the inner root sheath of whisker hair follicles, and spermatozoa). In addition, NNAT exhibited novel intracellular localization in acrosomes in the spermatozoa. Because the endoplasmic reticulum (ER) is excluded from spermatozoa and sarco/ER Ca 2+ -ATPase isoform 2 (SERCA2) is absent from the inner root sheath, these findings suggested that NNAT localization in the ER and its interaction with SERCA2 are cell- or tissue-specific properties.

This study aimed to describe the morphology, expression of IgA and IgG in adult yak tonsils. The 12 clinically healthy yak tonsils [3- to 6-year old, n = 12] were examined for morphology using light, and transmission electron microscopes. Expression of IgA and IgG was measured by qRT-PCR, ELISA, and immunohistochemistry. The results showed that the palatine tonsil, the tonsil of the soft palate, and the lingual tonsil were oropharyngeal tonsils. The stratified squamous epithelia covering them had a thick underlying layer of connective tissue and their crypts were heavily infiltrated by lymphocytes. The pharyngeal tonsil and the tubal tonsil were nasopharyngeal tonsils. The epithelia of them was predominantly pseudostratified columnar ciliary epithelium, which were loosely arranged with a number of desmosomes or intermediate junctions variably connecting them. The expression levels of IgA and IgG mRNA and protein from high to low was in the pharyngeal tonsil, palatine tonsil, tonsil of the soft palate, lingual tonsil, and tubal tonsil, respectively. Interestingly, the expression of IgG was very significantly higher than that of IgA in yak tonsils (P < 0.01). Both the IgA and IgG ASCs were distributed in the subepithelial areas of the non-reticular crypt epithelium, especially areas of pseudostratified columnar ciliary epithelium, the reticular crypt epithelium, lymphoid follicles, interfollicular areas, and with some of the positive cells aggregating around the glands. The results indicated that the tonsils were not only typical secondary lymphoid organs but also lymphoepithelial structures. IgG could be a significant component of mucosal immune responses in yak tonsils. Anat Rec, 302:999-1009, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The parakeratinized epithelium is a common and widespread type of keratinized epithelium in the oral cavity in adult birds. In contrast to orthokeratinized epithelium, which mostly covers mechanical papillae and the lingual nail, parakeratinized epithelium covers almost the entire dorsal surface of the tongue in birds. The characteristic feature of parakeratinized epithelium is the presence of nuclei in the keratinized layer. The present study aimed to investigate for the first time the micro- and ultrastructural changes of parakeratinized epithelium during embryonic development and to assess the readiness of the epithelium to serve protective functions during food transport to the esophagus. Three developmental stages were distinguished: embryonic, transformation, and pre-hatching stages. The embryonic stage lasts from the 9th to the 14th day of incubation and the epithelium is composed of undifferentiated epithelial cells. The transformation stage lasts from the 15th to the 22nd day of incubation and the epithelium undergoes transformation into stratified epithelium consisting of basal, intermediate, and superficial layers. The characteristic feature of this stage is formation of the periderm with osmophilic granules. The pre-hatching stage starts on the 23rd day, and the epithelium with a fully developed keratinized layer resembles that of the epithelium in adult animals. No periderm was observed on the epithelial surface. It was confirmed that at the time of hatching the parakeratinized epithelium is fully differentiated and ready to fulfill its function during food transport. The presence of periderm is a common feature characteristic for para- and orthokeratinized epithelium in the oral cavity of birds. However, the formation of the keratinized/cornified layer is different for these two types of keratinized epithelia.

Oral cancer has a high annual incidence rate all over the world, and the tongue is the most frequently affected anatomic structure. The current standard care is ablative surgery of malignant neoplasm, followed by tongue reconstruction with free flap. However, such reconstructive modalities with postsurgery radiotherapy or chemotherapy can hardly support the functional recovery of the tongue-particularly, functional taste bud regeneration-in reconstructed areas, thus seriously affecting patients' prognosis and life quality. Using a critical-sized tongue defect model in rats, we show that combinatory transplantation of small intestinal submucosa-extracellular matrix (SIS-ECM) with gingival mesenchymal stem cells (GMSCs) or their derivative exosomes promoted tongue lingual papillae recovery and taste bud regeneration as evidenced by increased expression of CK14, CK8, and markers for type I, II, and III taste bud cells (NTPdase 2, PLC-β2, and AADC, respectively). In addition, our results indicate that GMSCs or their derivative exosomes could increase BDNF expression, a growth factor that plays an important role in the proliferation and differentiation of epithelial basal progenitor cells into taste bud cells. Meanwhile, we showed an elevated expression level of Shh-which is essential for development, homeostasis, and maintenance of the taste bud organ-in wounded areas of the tongue among animals treated with GMSC/SIS-ECM or exosome/SIS-ECM as compared with SIS-ECM control. Moreover, our data show that GMSCs or their derivative exosomes promoted innervation of regenerated taste buds, as evidenced by elevated expressions of neurofilament and P2X 3 at the injury areas. Together, our findings indicate that GMSC/SIS-ECM and exosome/SIS-ECM constructs can facilitate taste bud regeneration and reinnervation with promising potential application in postsurgery tongue reconstruction of patients with tongue cancer.

A novel lingual epithelial organoid culture system using a three-dimensional (3D) matrix and growth factors has recently been established. In the culture system, organoids with multilayered squamous keratinized epithelium and typical morphological features of filiform papillae are generated from single lingual epithelial cells at a high efficiency. The culture system is created in order to observe the differentiation and maturation process of each lingual epithelial stem cell and to observe abnormal organoid formation from malignant cells obtained from carcinogen-treated mice. Thus, our culture system will contribute to the advancement of research into the regulatory mechanism of lingual epithelium and the underlying mechanisms of carcinogenesis.

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

This study was conducted with the aim of determining the morphology, distribution and heterogeneity of mast cells in the tongues of seagull (Larus fuscus), common buzzard (Buteo buteo), goose (Anser anser), white stork (Ciconia ciconia) and Gerze rooster. The study used five samples of tongue material from each of the healthy adult avian species. The samples were fixed in 10% neutral-buffered formalin (NBF) solution, then, after routine tissue follow-up, the samples blocked with paraplast. Cross-sections with 5-6 μm of thickness were stained with the 0.5% toluidine blue and alcian blue/safranin O (AB/SO). In all five avian species, it was found that the mast cells were in different sizes and round, oval or spindle-shaped based on their place of distribution. Mast cell numbers were determined in stained with toluidine blue, examined ×40 objectives in a 1 mm 2 area. It was observed that mast cell density in subepithelial lamina propria and microscopic papilla was higher in the tongues of all species. Mast cell distribution and heterogeneity varied through the tongue, and there were more mast cells in the dorsal side of the tongue than the ventral side. The highest amount of mast cells was found in the tongue of the Gerze rooster among all five species. In the tongue cross-sections stained with the combined method of alcian blue/safranin O (AB/SO), the mast cells were stained as AB (+), SO (+) and AB/SO (+) (mixed)., (© 2018 Blackwell Verlag GmbH.)

This research aimed to reveal the general morphology and topographic distribution of lingual papillae, epithelial characteristics, mucosal structure, and glands with their mucin content in the sheep tongue, with consideration of species-specific characteristics. The tongues of ten sheep were analyzed for this purpose. Filiform and fungiform papillae existed within the borders of the ventral surface of the lingual apex. The majority of the filiform papillae had multiple secondary projections. Fungiform papillae were also seen on the lingual torus among lenticular papillae, as well as 6 to 10 circumvallate papillae arranged on its caudal border. The species-specific details of the general anatomical structure of the tongue were determined and, in general, the papillary organization in the sheep was similar to goats, while the papillary organization also was similar to features with deer species, specifically the filiform papilla from the mechanical papillae and fungiform papilla from the gustatory papillae. Neutral and weak sulfated mucins and N-acetyl sialomucins were located in seromucous glands, salivary duct epithelium and von Ebner's glands. Carboxylated acid mucins and N-acetyl sialomucins were not present in seromucous and von Ebner's glands. In seromucous glands, MUC1, MUC5AC, and MUC6 localized only in epithelial cells of ducts, whereas MUC2 localized in both glandular and ductal epithelial cells. All MUCs were present in both von Ebner's glands and salivary ducts. We showed that this mucin composition, may serve as a physical barrier in the initial section of the digestive system. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Aim: The morphologic mechanism of orgasmic sensation has not yet been understood. Taste roseas may be stimulated by fructose via pudendal nerves, which has not been studied yet., Methods: In this study, 27 male adult rabbits were used, which were divided into three groups: 5 as control; 5 as SHAM and 17 used as study group. We injected 0.2 cc of distilled water to SHAM and 0.2 cc of fructose solution to the study group of their urethral orifices, and examined the occurrence of penile erection. The relationship between erection and pudendal nerve ganglia and penile tissues was statistically compared., Results: In animals with high neuron density of pudendal ganglia, more erection phenomenon was observed than those animals with low neuron density. Interestingly, neuron density of pudendal ganglia was 9.243 ± 542 /mm 3 in hypoactive and was 5.980 ± 463 /mm 3 in non-active animals (p < 0.05)., Conclusions: The seminal fructose may stimulate taste roseas of the urethra and glans penis via pudendal nerves. The present study describes a new neuro-morpho-chemical mechanism of orgasmic sensation with its neurosurgical aspect.

The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αβ and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1β, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa., Competing Interests: The authors have declared that no competing interests exist.

Salivary dysfunction commonly occurs in many older adults and is considered a physiological phenomenon. However, the genetic changes in salivary glands during aging have not been characterized. The present study analyzed the gene expression profile in salivary glands from accelerated aging klotho deficient mice (klotho-/-, 4 weeks old). Microarray analysis showed that 195 genes were differentially expressed (z-score > 2 in two independent arrays) in klotho null mice compared to wild-type mice. Importantly, alpha2-Na + /K + -ATPase (Atp1a2), Ca 2+ -ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to be regulators of submandibular salivary gland function, were significantly decreased. When a network was constructed from the differentially expressed genes, proliferator-activated receptor-γ (PPAR γ), which regulates energy homeostasis and insulin sensitivity, was located at the core of the network. In addition, the expression of genes proposed to regulate various PPAR γ-related cellular pathways, such as Klk1b26, Egfbp2, Cox8b, Gpx3, Fabp3, EGF, and NGFβ, was altered in the submandibular salivary glands of klotho-/- mice. Our results may provide clues for the identification of novel genes involved in salivary gland dysfunction. Further characterization of these differentially expressed genes will be useful in elucidating the genetic basis of aging-related changes in the submandibular salivary gland., (© 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

Tongue squamous epithelial cells are the main component of tongue coating, with proliferation, differentiation and apoptosis being the root cause of the formation and maintenance of tongue coating. The present study aimed to explore the molecular mechanism by which serum influences tongue coating, to enable a better understanding for future investigations. Tongue carcinoma squamous cells were exposed to serum‑starvation in vitro. Cellular proliferation and apoptosis were observed by using 3‑[4,5‑dimethyl‑2‑thiazolyl]‑2,5‑diphenyl‑2‑H‑tetrazolium bromide (MTT) assay, flow cytometry, Hoechst staining, scanning electron microscope (SEM), transmission electron microscope (TEM), and by measuring the expression ratio of B‑cell lymphoma 2 apoptosis regulator (Bcl‑2)/Bcl‑2 associated protein X apoptosis regulator (Bax) mRNA and protein by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting, respectively. MTT assays revealed that serum‑starvation results in suppression of cellular proliferation, while flow cytometry data revealed that serum‑starvation induces cell cycle arrest at G1 phase and increases apoptosis. In addition, chromatin condensation and membrane blebbing were observed through Hoechst staining, TEM and SEM. The Bcl‑2/Bax ratio was found to be significantly decreased in cells that had undergone serum‑starvation by both RT‑qPCR and western blotting analysis, further indicating that serum‑starvation induces apoptosis. Therefore, tongue carcinoma squamous cells in a serum‑free medium can simulate apoptosis related to the formation of tongue coating, which may offer guidance for future investigations about other factors.

The major salivary glands of birds develop by branching or elongation of the epithelial cords. The development of the minor salivary glands in form of the lingual glands has never been described. Among birds, only Anatidae have three types of the lingual glands: rostral, caudo-lateral, and caudo-medial lingual glands. The study aims to characterize the manner and rate of the lingual glands development in the domestic duck and their topographical arrangement relative to the hyoid apparatus. The study reveals that all three types of the lingual glands develop by branching. We describe five stages of the lingual glands development in the domestic ducks: prebud, initial bud, pseudoglandular, canalicular, and terminal bud stage. The pattern of the lingual glands development in birds is similar to that described for mammals, with the exception, that the terminal buds are formed at the same time as the lumen of the glands. Generally, the rostral lingual gland starts to branch earlier than the caudal lingual glands. The 3D-reconstruction shows the location and direction of lingual gland development relative to the entoglossal cartilage and basibranchial bone. Light microscopy and scanning electron microscopy allow to characterize the histogenesis of the embryonic epithelium into glandular epithelium. At a time of hatching only secretory units of caudal lingual glands resemble the secretory units of the adult domestic duck. The rostral and caudo-lateral lingual glands are arranged on the sides of the entoglossal cartilage and basibranchial bone and caudo-madial lingual glands are located over the basibranchial bone. We suggest that such an arrangement of the lingual glands in the domestic duck is important during food intake and responsible for reduction of friction and formation of food bites., (© 2017 Wiley Periodicals, Inc.)

Mouse taste receptor cells survive from 3-24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging.

Objective: To provide histological evidence to investigate a theory for post-tonsillectomy secondary hemorrhage (PTH) in a mouse model and to evaluate the potential for heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment on wound healing in this model., Methods: A prospective randomized single-blinded cohort study. A uniform tongue wound was created in 84 mice (day 0). Mice were randomized to HB-EGF (treatment, n = 42) or saline (control, n = 42). In treatment mice, HB-EGF 5 µg/ml was administered intramuscularly into the wound daily (days 0-14). In control mice, normal saline was administered daily. Three mice from each group were sacrificed daily through day 14 and the wounds evaluated histologically by blinded reviewers., Results: Key stages of wound healing, including keratinocyte proliferation and migration, wound contraction, epithelial separation, and neoangiogenesis, are defined with implications for post-tonsillectomy wound healing. Epithelial separation (59 vs. 100%, p = 0.003) and wound reopening (8 vs. 48%, p < 0.001) were reduced with HB-EGF. Epithelial thickness (220 vs. 30 µm, p = 0.04) was greater with HB-EGF. Wound closure (days 4-5 vs. day 6, p = 0.01) occurred earlier with HB-EGF., Conclusions: In healing of oral keratinocytes on muscle epithelial separation secondary to muscle, contraction occurs concurrently with neoangiogenesis in the base of the wound, increasing the risk of hemorrhage. This potentially explains why post-tonsillectomy secondary hemorrhage occurs and its timing. HB-EGF-treated wounds showed greater epithelial thickness, less frequent epithelial separation and wound reopening, and earlier wound closure prior to neovascularization, suggesting that HB-EGF may be a potential preventative therapy for PTH., Level of Evidence: NA-animal studies or basic research.

How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog ( Shh ), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh -expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration., Competing Interests: The authors declare no conflict of interest.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9 ki/ki mice). We show that DPP9 ki/ki mice die within 12-18h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9 ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9 ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors., (Copyright © 2017. Published by Elsevier Inc.)

The tongue of anteaters (Xenarthra, Pilosa, Vermilingua) is a highly specialized for myrmecophagy. Here, we describe the topography and histology of the tongue, and compare it to that of other xenarthrans and other myrmecophagous eutherian mammals. The tongue of Vermilingua is long and slender, with an apical protuberance, which differs between Myrmecophagidae and Cyclopes didactylus. In the former, the rostral region is conical, and in the latter, it is dorsoventrally compressed, as observed in sloths. The tongue of Vermilingua has filiform and circumvallate papillae on the surface; foliate and fungiform papillae are absent. The filiform papillae of Myrmecophaga tridactyla are simple all over the tongue, differing from Tamandua tetradactyla and Cyclopes didactylus, which present composed filiform papillae in the rostral and middle regions. Histologically, the tongue has a peculiar organization of muscular and neurovascular tissues, differing from the usual mammalian pattern. However, the tongue structure is less divergent in Cyclopes. The presence of two circumvallate papillae is common to the three major clades of Xenarthra (Cingulata, Folivora and Vermilingua). In each group, the tongue may reflect functional features related to myrmecophagous (anteaters and some armadillos), omnivorous (remaining armadillos) and folivorous (sloths) feeding habits. The similarities between the tongues of Vermiligua and other non-xenarthran eutherian myrmecophagous mammals are somewhat general and, under close inspection, superficial, being an example of different lineages achieving the same morphofunctional adaptations through distinct evolutionary pathways., (© 2017 Wiley Periodicals, Inc.)

Background: A comparative characterization of the oral mucosa in various animals is needed to identify the best animal model(s) for nonclinical evaluation of sublingual immunotherapy products. With this aim, we studied the histological characteristics and immune cell infiltrates of oral mucosae from common animal species., Methods: Three oral regions (i.e. ventral surface of the tongue, mouth floor and cheek) obtained from eight animal species, including rodents (i.e. mice, rats, hamsters, guinea pigs) and non-rodents (i.e. rabbits, dogs, minipigs and monkeys) were characterized by histology and immunohistology in comparison with a human tongue., Results: Rodents exhibit a thin keratinized epithelium with low epithelial extensions, whereas non-rodents, most particularly minipigs and monkeys, display a non-keratinized epithelium with larger rete ridges, similarly to humans. Glycogen-rich cells in the superficial epithelial layers are observed in samples from both minipigs, monkeys and humans. Comparable immune subpopulations detected in the 3 oral regions from rodent and non-rodent species include MHC-II+ antigen presenting cells, mostly CD163+ macrophages, located in the lamina propria (LP) and muscle tissue in the vicinity of resident CD3+CD4+ T cells. Limited numbers of mast cells are also detected in the LP and muscle tissue from all species., Conclusion: The oral mucosae of minipigs and monkeys are closest to that of humans, and the immune networks are quite similar between all rodents and non-rodents. Taking into account the ethical and logistical difficulties of performing research in the latter species, rodents and especially mice, should preferentially be used for pharmacodynamics/efficacy studies. Our data also support the use of minipigs to perform biodistribution and safety studies of sublingual immunotherapy products.

RNA-Seq is a powerful tool in transcriptomic profiling of cells and tissues. We recently identified many more taste buds than previously appreciated in chickens using molecular markers to stain oral epithelial sheets of the palate, base of oral cavity, and posterior tongue. In this study, RNA-Seq was performed to understand the transcriptomic architecture of chicken gustatory tissues. Interestingly, taste sensation related genes and many more differentially expressed genes (DEGs) were found between the epithelium and mesenchyme in the base of oral cavity as compared to the palate and posterior tongue. Further RNA-Seq using specifically defined tissues of the base of oral cavity demonstrated that DEGs between gustatory (GE) and non-gustatory epithelium (NGE), and between GE and the underlying mesenchyme (GM) were enriched in multiple GO terms and KEGG pathways, including many biological processes. Well-known genes for taste sensation were highly expressed in the GE. Moreover, genes of signaling components important in organogenesis (Wnt, TGFβ/ BMP, FGF, Notch, SHH, Erbb) were differentially expressed between GE and GM. Combined with other features of chicken taste buds, e.g., uniquely patterned array and short turnover cycle, our data suggest that chicken gustatory tissue provides an ideal system for multidisciplinary studies, including organogenesis and regenerative medicine.

Objectives: To evaluate chromosomal damage and cytotoxicity in exfoliated buccal mucosa cells, cells of the tongue and epithelial gingival cells from adults following CBCT scan and to compare the sensitivity of the different exfoliated cells to a same dosage of ionizing radiation., Methods: The study included 46 healthy participants (median age 27 years; age range 23-42 years) who had a CBCT scan. Exfoliated mucosa cells were collected immediately before the CBCT scan and 10 days after. Cells were centrifuged, fixed in the fluid of methanol : glacial acetic acid (3 : 1) and stained using the method of Schiff's reagent and fast green. One observer analyzed all the slides. For interobserver variances, a second observer scored 16 slides chosen from all the subjects. The same set of 16 slides were analyzed once again a month later for intraobserver variances., Results: There is no significant differences for micronucleated cells before and after a CBCT scan in exfoliated buccal mucosa cells (p = 0.476), cells of the tongue (p = 0.884) and epithelial gingival cells (p = 0.362). The frequencies of pyknosis cell and karyolysis cell had significantly increased after CBCT scan in the three groups. No significant difference was found among the three kinds of mucosa cells (p = 0.557). The interobserver (p = 0.624) and intraobserver (p = 0.193) variances were not significant., Conclusions: A CBCT scan may induce cytotoxicity but not chromosomal damage in the oral mucosa cells, including buccal mucosa cells, cells of the tongue or epithelial gingival cells. The sensitivity of the different exfoliated cells to the same dosage of radiation had no statistically significant difference.

In the oral cavity, the tongue is the anatomic subsite most commonly involved by invasive squamous cell carcinoma. Current treatment protocols often require significant tissue resection to achieve adequate negative margins and optimal local tumor control. Reconstruction of the tongue while preserving and/or restoring its critical vocal, chewing, and swallowing functions remains one of the major challenges in head and neck oncologic surgery. We investigated the in vitro feasibility of fabricating a novel combinatorial construct using porcine small intestinal submucosa extracellular matrix (SIS-ECM) and human gingiva-derived mesenchymal stem cells (GMSCs) as a GMSC/SIS-ECM tissue graft for the tongue reconstruction. We developed a rat model of critical-sized myomucosal defect of the tongue that allowed the testing of therapeutic effects of an acellular SIS-ECM construct versus a GMSC/SIS-ECM construct on repair and regeneration of the tongue defect. We showed that the GMSC/SIS-ECM construct engrafted at the host recipient site, promoted soft tissue healing, and regenerated the muscular layer, compared to the SIS-ECM alone or nontreated defect controls. Furthermore, our results revealed that transplantation of the GMSC/SIS-ECM construct significantly increased the expression of several myogenic transcriptional factors and simultaneously suppressed the expression of type I collagen at the wounded area of the tongue. These compelling findings suggest that, unlike the tongue contracture and fibrosis of the nontreated defect group, transplantation of the combinatorial GMSC/SIS-ECM constructs accelerates wound healing and muscle regeneration and maintains the overall tongue shape, possibly by both enhancing the function of endogenous skeletal progenitor cells and suppressing fibrosis. Together, our findings indicate that GMSC/SIS-ECM potentially served as a myomucosal graft for tongue reconstruction postsurgery of head and neck cancer.

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been shown to have anti-proliferative, anti-angiogenic, and proapoptotic effects, leading to interest in their use as cancer therapeutics. Pioglitazone, a U.S. Food and Drug Administration-approved type II diabetes medication and PPARγ agonist, may have a role in adjuvant head-and-neck squamous cell carcinoma treatment or prevention. Therefore, the purpose of this study was: 1) to treat oral cavity cancer cells with the PPARγ activator, pioglitazone, to analyze gene expression changes; and 2) to compare those changes with our preexisting genomic data for development of hypothesis-driven additional basic and clinical studies., Study Design: Prospective in vitro., Methods: We utilized microarray technology, as well as OCPlus (Bioconductor open source software) and Ingenuity Pathway Analysis (Qiagen, Redwood City, CA), to analyze differential gene expression in tumor and pioglitazone-treated tumor cells on a genome-wide level to demonstrate the feasibility of such an approach and determine appropriate sample size for future investigations., Results: We found that approximately 35 samples are required to adequately power future studies. We next discovered that pioglitazone significantly affects Inducible T-Cell Costimulator (iCOS)-Ligand for the T-cell-specific cell surface receptor ICOS (iCOSL) and type II diabetes mellitus pathways as a putative anti-cancer mechanism., Conclusion: Genome-wide analysis is possible for the exploration of differential pathway modulation and rapid hypothesis generation. Both inflammation and type II diabetes pathways were significantly altered and therefore might provide unique hypothesis-driven pharmacodynamic parameters for future in vitro or in vivo studies utilizing thiazolidinediones. These techniques could be applied to microarray or other high throughput data from a variety of hypothesis-generating research scenarios in otolaryngology (e.g., middle ear proteomics, sinus microbiome studies)., Level of Evidence: NA. Laryngoscope, 127:E124-E131, 2017., (© 2016 The American Laryngological, Rhinological and Otological Society, Inc.)

TRPC (transient receptor potential cation channel subfamily C) members are nonselective monovalent cation channels and control Ca2+ inflow. In this study, immunohistochemistry for TRPC1, TRPC3, and TRPC4 was performed on rat oral and craniofacial structures to elucidate their distribution and function in the peripheries. In the trigeminal ganglion (TG), 56.1, 84.1, and 68.3% of sensory neurons were immunoreactive (IR) for TRPC1, TRPC3, and TRPC4, respectively. A double immunofluorescence method revealed that small to medium-sized TG neurons co-expressed TRPCs and calcitonin gene-related peptide. In the superior cervical ganglion, all sympathetic neurons showed TRPC1 and TRPC3 immunoreactivity. Parasympathetic neurons in the submandibular ganglion, tongue, and parotid gland were TRPC1, TRPC3, and TRPC4 IR. Gustatory and olfactory cells were also IR for TRPC1, TRPC3, and/or TRPC4. In the musculature, motor endplates expressed TRPC1 and TRPC4 immunoreactivity. It is likely that TRPCs are associated with sensory, autonomic, and motor functions in oral and craniofacial structures., (© 2017 S. Karger AG, Basel.)

Few reports have been published on the early microbiota in infants with various types of cleft palate. We assessed the formation of the oral microbiota in infants with complete cleft lip and palate (CLP n = 30) or cleft soft palate (CSP n = 25) in the neonatal period (T1 time) and again in the gum pad stage (T2 time). Culture swabs from the tongue, palate, and/or cleft margin at T1 and T2 were taken. We analysed the prevalence of the given bacterial species (the percentage) and the proportions in which the palate and tongue were colonised by each microorganism. At T1, Streptococcus mitis (S. mitis) were the most frequently detected in subjects with CLP or CSP (63% and 60%, resp.). A significantly higher frequency of methicillin-sensitive Staphylococcus aureus ( S. aureus MSSA) was observed in CLP compared to the CSP group. At T2, significantly higher percentages of S. mitis , S. aureus MSSA, Staphylococcus epidermidis , and members of the Enterobacteriaceae family were noted in CLP infants compared to the CSP. S. mitis and Streptococcus sanguinis appeared with the greatest frequency on the tongue, whereas Streptococcus salivarius was predominant on the palate. The development of the microbiota in CLP subjects was characterised by a significant increase in the prevalence of pathogenic bacteria.

Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host β-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/β-actin and IRES/β-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1μl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/β-actin test and 97% (95% CI; 87-100%) for the IRES/β-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Data from diffusion magnetic resonance imaging (dMRI) can be used to reconstruct fiber tracts, for example, in muscle and white matter. Estimation of fiber orientations (FOs) is a crucial step in the reconstruction process and these estimates can be corrupted by noise. In this paper, a new method called Fiber Orientation Reconstruction using Neighborhood Information (FORNI) is described and shown to reduce the effects of noise and improve FO estimation performance by incorporating spatial consistency. FORNI uses a fixed tensor basis to model the diffusion weighted signals, which has the advantage of providing an explicit relationship between the basis vectors and the FOs. FO spatial coherence is encouraged using weighted ℓ1-norm regularization terms, which contain the interaction of directional information between neighbor voxels. Data fidelity is encouraged using a squared error between the observed and reconstructed diffusion weighted signals. After appropriate weighting of these competing objectives, the resulting objective function is minimized using a block coordinate descent algorithm, and a straightforward parallelization strategy is used to speed up processing. Experiments were performed on a digital crossing phantom, ex vivo tongue dMRI data, and in vivo brain dMRI data for both qualitative and quantitative evaluation. The results demonstrate that FORNI improves the quality of FO estimation over other state of the art algorithms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The hypoglossal motor nucleus is one of the efferent components of the neural network underlying the tongue prehension behavior of Ranid frogs. Although the appropriate pattern of the motor activity is determined by motor pattern generators, sensory inputs can modify the ongoing motor execution. Combination of fluorescent tracers were applied to investigate whether there are direct contacts between the afferent fibers of the trigeminal, facial, vestibular, glossopharyngeal-vagal, hypoglossal, second cervical spinal nerves and the hypoglossal motoneurons. Using confocal laser scanning microscope, we detected different number of close contacts from various sensory fibers, which were distributed unequally between the motoneurons innervating the protractor, retractor and inner muscles of the tongue. Based on the highest number of contacts and their closest location to the perikaryon, the glossopharyngeal-vagal nerves can exert the strongest effect on hypoglossal motoneurons and in agreement with earlier physiological results, they influence the protraction of the tongue. The second largest number of close appositions was provided by the hypoglossal and second cervical spinal afferents and they were located mostly on the proximal and middle parts of the dendrites of retractor motoneurons. Due to their small number and distal location, the trigeminal and vestibular terminals seem to have minor effects on direct activation of the hypoglossal motoneurons. We concluded that direct contacts between primary afferent terminals and hypoglossal motoneurons provide one of the possible morphological substrates of very quick feedback and feedforward modulation of the motor program during various stages of prey-catching behavior.

As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.