Rapid Kinetic Interactions of Sugar and Sugar Alcohol with Sweet Taste Receptors on Live Cells Using Stopped-Flow Spectroscopy.

The subjective measurement of the dynamic perception of sweetness is a problem in food science. Herein, the rapid interactions of sugars and sugar alcohols with sweet taste receptors on living cells on a millisecond timescale were studied via stopped-flow fluorescence spectroscopy. According to the rapid-kinetic parameters, sweeteners were divided into two groups. Sweeteners in group I disrupted the hydrogen bond network structure of water, and the apparent rate constant (kobs) was in the range of 0.45-0.6 s-1. Sweeteners in group II promoted the hydrogen bond formation of water, and the kobs was mostly in the range of 0.6-0.75 s-1. For most sweeteners, the kobs of cell responses was negatively correlated with the apparent specific volume of sweeteners. The differences in the cellular responses may be attributed to the disturbance in the water structure. Experimental results showed that the kinetic parameters of sweet cell responses reflected the dynamic perception of sweetness. Rapid kinetics, solution thermodynamic analysis, and water structure analysis enriched the physicochemical study of the sweetness mechanism and can be used to objectively evaluate the dynamic perception of sweetness.

Crypt-like patterned electrospun nanofibrous membrane and probiotics promote intestinal epithelium models close to tissues.

In vitro intestinal epithelium models have drawn great attention to investigating intestinal biology in recent years. However, the difficulty to maintain the normal physiological status of primary intestinal epithelium in vitro limits the applications. Here, we designed patterned electrospun polylactic acid (PLA) nanofibrous membranes with crypt-like topography and mimic ECM fibrous network to support crypt culture and construct in vitro intestinal epithelium models. The patterned electrospun PLA nanofibrous membranes modified with Matrigels at 0 °C showed high biocompatibility and promoted cell growth and proliferation. The constructed duodenum epithelium models and colon epithelium models on the patterned electrospun PLA nanofibrous membranes expressed the typical differentiation markers of intestinal epithelia and the gene expression levels were close to the original tissues, especially with the help of probiotics. The constructed intestinal epithelium models could be used to assess probiotic adhesion and colonization, which were verified to show significant differences with the Caco-2 cell models due to the different cell types. These findings provide new insights and a better understanding of the roles of biophysical, biochemical, and biological signals in the construction of in vitro intestinal epithelium models as well as the potential applications of these models in the study of host-gut microbes interactions. KEY POINTS: • Patterned electrospun scaffold has crypt-like topography and ECM nanofibrous network. • Matrigels at 0°C modify scaffolds more effectively than at 37°C. • Synergy of biomimic scaffold and probiotics makes in vitro model close to tissue.

Intestinal models based on biomimetic scaffolds with an ECM micro-architecture and intestinal macro-elasticity: close to intestinal tissue and immune response analysis.

The synergetic biological effect of scaffolds with biomimetic properties including the ECM micro-architecture and intestinal macro-mechanical properties on intestinal models in vitro remains unclear. Here, we investigate the profitable role of biomimetic scaffolds on 3D intestinal epithelium models. Gelatin/bacterial cellulose nanofiber composite scaffolds crosslinked by the Maillard reaction are tuned to mimic the chemical component, nanofibrous network, and crypt architecture of intestinal ECM collagen and the stability and mechanical properties of intestinal tissue. In particular, scaffolds with comparable elasticity and viscoelasticity of intestinal tissue possess the highest biocompatibility and best cell proliferation and differentiation ability, which makes the intestinal epithelium models closest to their counterpart intestinal tissues. The constructed duodenal epithelium models and colon epithelium models are utilized to assess the immunobiotics-host interactions, and both of them can sensitively respond to foreign microorganisms, but the secretion levels of cytokines are intestinal cell specific. The results demonstrate that probiotics alleviate the inflammation and cell apoptosis induced by Escherichia coli, indicating that probiotics can protect the intestinal epithelium from damage by inhibiting the adhesion and invasion of E. coli to intestinal cells. The designed biomimetic scaffolds can serve as powerful tools to construct in vitro intestinal epithelium models, providing a convenient platform to screen intestinal anti-inflammatory components and even to assess other physiological functions of the intestine.

[Genetic Analysis and Prenatal Diagnosis of a Family with Hereditary Spherocytosis Caused by a Novel Compound Heterozygous Mutation of SPTB Gene].

OBJECTIVE: To investigate the clinical and genetic characteristics of a family with hereditary spherocytosis (HS), to clarify the cause of the disease, and to provide the basis for genetic counseling and prenatal diagnosis. METHODS: The clinical data of proband and his parents were collected, and HS-related pathogenic genovariation of the proband was detected by high throughput sequencing. Suspected pathogenic mutation sites were verified by PCR-Sanger sequencing, and the fetus were conceived by a proband mother underwent prenatal diagnosis. RESULTS: Clinical manifestations of the proband showed moderate anemia, mild splenomegaly, and jaundice (an indirect increase of bilirubin). The gene detection showed that the proband showed compound heterozygous mutations of SPTB gene c. 6095T > C (p.Leu2032Pro) and c. 6224A > G (p.Glu2075Gly), which was inherited from the asymptomatic mother and father, respectively. Both mutations were detected rarely in the common population. Prenatal diagnosis revealed that the fetus inherited a mutant gene of the mother. CONCLUSION: The compound heterozygous mutations of SPTB genes c.6095T>C (p.Leu2032Pro) and c.6224A>G (p.Glu2075Gly) were the causes of the family disease, which provides a basis for family genetic counseling and prenatal diagnosis. This report is the first one found in the HGMD,1000G and EXAC database, which provides an addition to the mutation profile of the SPTB gene.

Histone deacetylase 6 promotes skin wound healing by regulating fibroblast migration and differentiation in aged mice.

Skin wound healing tends to slow down with aging, which is detrimental to both minor wound recovery in daily life and the recovery after surgery. The aim of current study was to explore the effect of histone deacetylase 6 (HDAC6) on wound healing during aging. Cultured human dermal fibroblasts (HDFs) and mouse full-thickness skin wound model were used to explore the functional changes of replicative senescent dermal fibroblasts and the effect of aging on skin wound healing. Scratch wound healing assay revealed significantly decreased migration speed of senescent HDFs, and BrdU incorporation assay indicated their considerably retardant proliferation. The protein expression levels of collagen and HDAC6 were significantly decreased in both senescent HDFs and skin tissues from aged mice. HDAC6 activity inhibition with highly selective inhibitor tubastatin A (TsA) or HDAC6 knockdown with siRNA decreased the migration speed of HDFs and considerably suppressed fibroblast differentiation induced by transforming growth factor-ß1 (TGF-ß1), which suggests the involvement of HDAC6 in regulating fundamental physiological activities of dermal fibroblasts. In vivo full-thickness skin wound healing was significantly delayed in young HDAC6 knockout mice when compared with young wild type mice. In addition, the wound healing was significantly slower in aged wild type mice than that in young wild type mice, and became even worse in aged HDAC6 knockout aged mice. Compared to the aged wild type mice, aged HDAC6 knockout mice exhibited delayed angiogenesis, reduced collagen synthesis, and decreased collagen deposition in skin wounds. Together, these results suggest that delayed skin wound healing in aged mice is associated with impaired fibroblast function. Adequate expression and activity of HDAC6 are required for fibroblasts migration and differentiation.

Sweet preference modified by early experience in mice and the related molecular modulations on the peripheral pathway.

The sweet taste is of immense interest to scientists and has been intensively studied during the last two decades. However, the sweet preference modification and the related mechanisms are still unclear. In this study, we try to establish a mice model with manipulated sweet taste preference and explore the involved possible molecular mechanisms. The animals were exposed to acesulfame-K via maternal milk during lactation and the sweet preference tests were carried out when they grew to adulthood. Our results showed that the preference thresholds for sweet taste were increased in adults by early acesulfame-K exposure and the preference ratios for sweet tastants at low or preferred concentrations were decreased. Moreover, by means of qRT-PCR and Western blot, we observed the increased expression of leptin receptor Ob-Rb and downregulation of Gα-gustducin protein in the soft palate. Thereby, the sweet taste sensitivity may be modified by early sweetener experience during lactation. Along the peripheral sweet sensory pathway, the sweet regulator receptors Ob-Rb, CB1 and components of sweet transduction signal Gα-gustducin and T1R2 in both the soft palate and tongue may be cooperatively involved in the plastic development of sweet taste.

Effects of early intraoral acesulfame-K stimulation to mice on the adult's sweet preference and the expression of α-gustducin in fungiform papilla.

Exposure to artificial sweetener acesulfame-K (AK) at early development stages may influence the adult sweet preference and the periphery gustatory system. We observed that the intraoral AK stimulation to mice from postnatal day 4 (P4) to weaning decreased the preference thresholds for AK and sucrose solutions in adulthood, with the preference pattern unchanged. The preference scores were increased in the exposure group significantly when compared with the control group at a range of concentrations for AK or sucrose solution. Meanwhile, more α-Gustducin-labeled fungiform taste buds and cells in a single taste bud were induced from week 7 by the early intraoral AK stimulation. However, the growth in the number of α-Gustducin-positive taste bud or positive cell number per taste bud occurred only in the anterior region, the rostral 1-mm part, but not in the intermediate region, the caudal 4-mm part, of the anterior two-third of the tongue containing fungiform papillae. This work extends our previous observations and provides new information about the developmental and regional expression pattern of α-Gustducin in mouse fungiform taste bud under early AK-stimulated conditions.

Effects of mother's dietary exposure to acesulfame-K in Pregnancy or lactation on the adult offspring's sweet preference.

This study investigates whether mother's exposure to the artificial sweetener acesulfame-K (AK) during pregnancy or lactation affected her adult offspring's sweet preference. It was found that mother's dietary exposure to AK in pregnancy or lactation decreased the preference thresholds for AK and sucrose solutions in the adult offspring, whereas the preference pattern and the most preferred concentration for AK or sucrose solution were unchanged. Furthermore, the preference scores in the exposure groups were increased significantly when compared with the control group at a range of concentrations for AK or sucrose solution. The existence of AK and its dynamic changes within 24 h in amniotic fluid during pregnancy or in mother's milk during lactation after a single oral infusion of AK solution were revealed by the methods of reversed-phase high-performance liquid chromatography and mass spectrometry. Our data suggest that AK can be ingested by the prenatal or postnatal mice through their mother's amniotic fluid or breast milk, producing a long-dated function on the adult's sweet preference.

Facilitation of the development of fungiform taste buds by early intraoral acesulfame-K stimulation to mice.

The gustatory system is susceptible to anatomical modification by postnatal taste stimulations. This study investigated the effects of early intraoral infusion of acesulfame-K solution on the development of fungiform taste buds in mice. It was found that the acesulfame-K infusion increased the number, promoted the maturation, and enlarged the size of taste bud during the postnatal stages, compared with the age-matched controls. This provides fundamental and new information about the development of taste bud under normal and early acesulfame-K-stimulated conditions.

A reliable method to obtain cells of taste buds from fungiform papillae of mice.

Taste buds consist of four kinds of cells which have distinct characteristics and play different roles in recognizing chemical compounds contained in foodstuffs. In this study we describe a procedure for separating viable taste bud cells from the fungiform papillae in mice. After sacrifice with CO(2), the mouse tongue was excised and immediately incubated in collagenase II and dispase II. The epithelium with fungiform papillae was then peeled away from underlying tissue and the anterior one-third region was incubated in a solution of 0.25% trypsin and 0.02M ethylene-diamine-tetraacetic acid (EDTA) for 8-12min. Following incubation, a cell suspension was obtained by mechanical dissociation. Cells in suspension were identified as taste bud cells by their morphology and by immunofluorescence. A 0.25% trypan blue staining demonstrated that nearly 90% of these cells remained viable. Micrographs from scanning electron microscopy illustrated that taste buds were dissociated from the fungiform papillae, while maintaining the integrity of the other part of the dissociated lingual epithelium during incubation. Such a method allows acquisition of viable taste cells and will aid further research in the study of gustatory characteristics.

The relationship between fungiform papillae density and detection threshold for sucrose in the young males.

The aim of this study was to investigate the relationship of fungiform papillae density with taste detection thresholds for sucrose of young male adults. One hundred and eighty two subjects aged 18-23 years (mean age: 21.9 +/- 1.2 years) were included. The densities of fungiform papillae were recorded with the aid of the digital camera, and the taste detection thresholds for sucrose were detected using a modified forced-choice triangle test. The mean density of papillae within all 170 statistic participants was 92.43 +/- 2.64/cm(2), for the 6-mm-diameter stained section of the tongue tip. The average detection threshold was 10.83 +/- 0.24 mmol/l, and the highest and lowest detection thresholds were 19.88 +/- 1.31 and 5.85 +/- 0.43 mmol/l, respectively. Also, an inverse correlation between the fungiform papillae density and the detection threshold was observed.

Quantitative study of taste bud distribution within the oral cavity of the postnatal mouse.

The aim of this study was to investigate the age-related developmental changes of taste bud distribution within the subpopulations at different postnatal ages in the mouse oral cavity. Developmental changes of taste bud distribution on the soft palate, fungiform, foliate and circumvallate papillae in the mouse oral cavity were examined histologically at different postnatal ages. After paraffin embedding, complete serial sections at 10mum thickness were made and stained by routine hematoxylin-eosin staining methods. Digitised images for each section were examined carefully. The existence of a taste pore was used to identify mature taste buds. A two-way analysis of variance (group versus age) was used to analyse differences in taste bud number and characteristics for each of the developmental changes. An independent measures t-test was used to compare two means. No taste buds with pores were observed at birth within circumvallate and foliate papillae. However, 61% of the circumvallate and 58% of the foliate taste buds contained taste pores at 2 weeks after birth. In contrast, at birth, 55% of the taste buds on the soft palate and only 22% of the taste buds within fungiform papillae contained taste pores. Then, the number of mature taste buds (taste buds with pores) increased rapidly 1 week after birth, resulting in 90% of soft palate taste buds and 32% of fungiform taste buds containing taste pores. These results suggests that the earlier maturation of soft palate taste buds compared with the other populations in the oral cavity raises evidence of their significant role in the taste mechanism, especially in the early life of the mouse.

Regional differences in taste bud distribution and alpha-gustducin expression patterns in the mouse fungiform papilla.

The regional differences between distribution patterns and alpha-gustducin expression patterns of the fungiform (FF) taste buds were investigated in the adult mouse, using hematoxylin-eosin staining and immunofluorescence histochemistry on the most anterior region of the tongue (the first millimeter) through the intermediate region of the tongue (the last 1-4 mm). Paraffin sections were prepared from the tip to posterior regions (anterior and intermediate region containing the FF taste buds) of the adult mouse tongue. Results indicate that there were significant regional differences in size and density of taste buds, the cell counts of the single taste bud, and the alpha-gustducin-immunoreactive taste buds between the 2 regions. The taste bud had a characteristic onion-like appearance, and the alpha-gustducin-immunoreactive cell was spindle shaped with elongated processes extending from the base to the pore of the taste buds. These results provide a detailed insight to better understand regional descriptions of mouse taste bud density and size and alpha-gustducin expression with the mouse model.

Differentiation of alpha-gustducin in taste buds of the mouse soft palate and fungiform papillae.

We used alpha-gustducin, a type II taste-cell-specific G protein, to investigate the onset of taste transduction and its relation to the development of the soft palate (SP) and fungiform (FF) papillae taste buds in the mouse. Paraffin wax embedded sections were prepared from the SP and anterior region of the tongue of the mouse from birth until postnatal day (PD) 63. No alpha-gustducin-immunoreactive cells were observed on the day of birth. One day later, alpha-gustducin was immunolocalised in taste buds with pores with a relatively higher frequency recorded in the SP as compared with the FF papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. On PD 7, the number of taste buds containing alpha-gustducin-immunoreactive cells in the SP was three times greater than that of FF papillae. Our results indicate that taste transduction is essentially acquired from the time of birth. Moreover, the onset of taste transduction by the SP taste buds developed earlier than that achieved by taste buds in the FF papillae.